WO2008021183A9 - Procédés d'identification, d'évaluation, et de traitement de patients soumis à une thérapie anticancéreuse - Google Patents

Procédés d'identification, d'évaluation, et de traitement de patients soumis à une thérapie anticancéreuse

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Publication number
WO2008021183A9
WO2008021183A9 PCT/US2007/017716 US2007017716W WO2008021183A9 WO 2008021183 A9 WO2008021183 A9 WO 2008021183A9 US 2007017716 W US2007017716 W US 2007017716W WO 2008021183 A9 WO2008021183 A9 WO 2008021183A9
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WO
WIPO (PCT)
Prior art keywords
markers
expression
patient
marker
predictive
Prior art date
Application number
PCT/US2007/017716
Other languages
English (en)
Other versions
WO2008021183A3 (fr
WO2008021183A2 (fr
Inventor
Barbara M Bryant
Andrew I Damokosh
George Mulligan
Original Assignee
Millennium Pharm Inc
Barbara M Bryant
Andrew I Damokosh
George Mulligan
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Millennium Pharm Inc, Barbara M Bryant, Andrew I Damokosh, George Mulligan filed Critical Millennium Pharm Inc
Priority to CA002660275A priority Critical patent/CA2660275A1/fr
Priority to JP2009523844A priority patent/JP5725711B2/ja
Priority to EP07836670A priority patent/EP2046973A4/fr
Priority to AU2007284724A priority patent/AU2007284724B2/en
Priority to MX2009001489A priority patent/MX2009001489A/es
Publication of WO2008021183A2 publication Critical patent/WO2008021183A2/fr
Publication of WO2008021183A9 publication Critical patent/WO2008021183A9/fr
Publication of WO2008021183A3 publication Critical patent/WO2008021183A3/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • cancer patients including, e.g., hematological cancer patients (e.g., multiple myeloma, leukemias, lymphoma, etc) as well as solid tumor cancer patients (e.g., lung, breast, prostate, ovary, colon, kidney, liver), who would benefit from particular cancer inhibition therapies as well as those who would benefit from a more aggressive and/or alternative cancer inhibition therapy, e.g., alternative to a cancer therapy or therapies the patient has received, thus resulting in appropriate preventative measures.
  • hematological cancer patients e.g., multiple myeloma, leukemias, lymphoma, etc
  • solid tumor cancer patients e.g., lung, breast, prostate, ovary, colon, kidney, liver
  • cancer inhibition therapies e.g., alternative to a cancer therapy or therapies the patient has received, thus resulting in appropriate preventative measures.
  • the proteasome is a multi-enzyme complex present in all cells which play a role in degradation of proteins involved in regulation of the cell cycle.
  • King et al demonstrated that the ubiquitin-proteasome pathway plays an essential role in regulating cell cycle, neoplastic growth and metastasis.
  • a number of key regulatory proteins, including p53, cyclins, and the cyclin- dependent kinases p21 and p27 ⁇ p ⁇ are temporally degraded during the cell cycle by the ubiquitin- proteasome pathway. The ordered degradation of these proteins is required for the cell to progress through the cell cycle and to undergo mitosis. See, e.g.. Science 274:1652-1659 (1996).
  • NF-kB the transcription factor NF-kB
  • DcB the inhibitor protein DcB
  • NF-kB plays a central role in the regulation of genes involved in the immune and inflammatory responses.
  • Read et al. demonstrated that the ubiquitin-proteasome pathway is required for expression of cell adhesion molecules, such as E-selectin, ICAM-I, and VCAM-I. See Immunity 2:493-506 (1995).
  • bortezomib N-pyrazinecarbonyl-L-phenylalanine-L-leucineboronic acid, PS-341) (VELCADE ® for injection, Millennium Pharmaceuticals, Inc., Cambridge, MA; Johnson & Johnson Pharmaceutical Research and Development L.L.C.) has been approved for treatment of relapsed multiple myeloma.
  • bortezomib N-pyrazinecarbonyl-L-phenylalanine-L-leucineboronic acid, PS-3411
  • VELCADE ® for injection
  • Millennium Pharmaceuticals, Inc. Cambridge, MA
  • Johnson & Johnson Pharmaceutical Research and Development L.L.C. Johnson & Johnson Pharmaceutical Research and Development
  • Bortezomib inhibits nuclear factor- ⁇ B (NF-KB) activation, attenuates interleukin-6 (IL-6) mediated cell growth, and has a direct apoptotic effect, and possibly an anti-angiogenic effect. Additionally, bortezomib is directly cytotoxic to myeloma cells in culture, independent of their p53 status. See, e.g., Hideshima T, et al. Cancer Res. 61:3071-6 (2001).
  • bortezomib In addition to a direct cytotoxic effect of bortezomib on myeloma cells, bortezomib inhibits tumor necrosis factor alpha (TNFoO stimulated intercellular adhesion molecule-1 (ICAM-I) expression by myeloma cells and ICAM-I and vascular cell adhesion molecule-1 (VCAM-I) expression on bone marrow stromal cells (BMSCs), resulting in decreased adherence of myeloma cells and, consequently, in decreased cytokine secretion.
  • TNFoO stimulated intercellular adhesion molecule-1 (ICAM-I) expression by myeloma cells and ICAM-I and vascular cell adhesion molecule-1 (VCAM-I) expression on bone marrow stromal cells (BMSCs) resulting in decreased adherence of myeloma cells and, consequently, in decreased cytokine secretion.
  • bortezomib By inhibiting interactions of myeloma cells with the surrounding bone marrow, bortezomib can inhibit tumor growth and survival, as well as angiogenesis and tumor cell migration.
  • the antineoplastic effect of bortezomib may involve several distinct mechanisms, including inhibition of cell growth signaling pathways, dysregulation of the cell cycle, induction of apoptosis, and inhibition of cellular adhesion molecule expression.
  • bortezomib induces apoptosis in cells that over express B-cell lymphoma 2 (Bcl-2), a genetic trait that confers unregulated growth and resistance to conventional chemotherapeutics. McConkey DJ, et al. The proteasome as a new drug target in metastatic prostate cancer.
  • Glucocorticoidal steroids are capable of causing apoptotic death of many varieties of cells, and a selection of glucocorticoidal steroids have consequently been used in the treatment of various malignancies, including lymphoid malignancies, and combination therapies in solid tumors.
  • various malignancies including lymphoid malignancies, and combination therapies in solid tumors.
  • the optimal therapy for relapsed myeloma is not established, but high- dose dexamethasone is commonly used. See, e.g., Kumar A, et al. Lancet Oncol; 4:293-304 (2003); Alexanian R, et al. Ann Intern Med. 105:8-11 (1986); Friedenberg WR, et al.
  • corticosteroids have demonstrated use in cancer treatments, including hydrocortisone in combination therapy for prostate cancer, predisolone in leukemia, prednisolone in lymphoma treatment, and triamcinolone has recently demonstrated some anti-cancer activity. See, e.g., Scholz M., et al., J. Urol. 173:1947-52.(2005); Sano J., et al, Res Vet ScL (May 10, 005); Zinzani PL. et a ⁇ .,Semin Oncol. 32(1 Suppl l):S4-10.
  • the present invention is based, in part, on the identification of individual markers and marker sets that can be used to determine whether enhanced survival time can be expected by treatment with a proteasome inhibition therapy and/or a glucocorticoid therapy or whether an alternative therapy to and/or a more aggressive therapy with a proteasome inhibitor and/or glucocorticoid inhibitor may enhance expected survival time.
  • the compositions and methods provided herein can be used to determine whether a patient is expected to be a long term or short term survivor to a proteasome inhibition therapeutic agent or a proteosome inhibitor dosing or administration regimen.
  • compositions and methods provided herein can be used to determine whether a patient is expected to be a long term or short term survivor to a glucocorticoid therapeutic agent or a glucocorticoid dosing or administration regimen. Based on these identifications, the present invention provides, without limitation: 1) methods and compositions for determining whether a proteasome inhibition therapy regimen and/or a glucocorticoid therapy regimen will or will not be effective to enhance patient survival time; 2) methods and compositions for monitoring the effectiveness of a proteasome inhibition therapy (a proteasome inhibitor agent or a combination of agents) and/or a glucocorticoid therapy (a glucocorticoid agent or combination of agents) and dosing and administrations used for the treatment of tumors; 3) methods and compositions for treatments of tumors comprising, e.g., proteasome inhibition therapy regimen and/or glucocorticoid therapy regimen; and 4) methods and compositions for identifying specific therapeutic agents and combinations of therapeutic agents
  • the markers of the present invention whose expression are predictive of short term and long term survival after treatment with a proteosome inhibitor and/or glucocorticoid inhibitor, are identified in Table 1 and Table 2.
  • a tumor By examining the expression of one or more of the identified markers or marker sets in a tumor, it is possible to determine which therapeutic agent, combination of agents, dosing and/or administration regimen is expected to enhance survival time.
  • a cancer By examining the expression of one or more of the identified markers or marker sets in a cancer, it is also possible to determine which therapeutic agent, combination of agents, dosing and/or administration regimen is less likely to enhance survival time.
  • By examining the expression of one or more of the identified markers or marker sets it is therefore possible to eliminate ineffective or inappropriate therapeutic agents.
  • these determinations can be made on a patient by patient basis. Thus, one can determine whether or not a particular therapeutic regimen is likely to benefit a particular patient or type of patient, and/or whether a particular regimen should be started or avoided, continued, discontinued or altered.
  • the present invention is directed to methods of identifying and/or selecting a cancer patient who is expected to demonstrate enhanced survival to a therapeutic regimen, e.g., as compared to a patient identified as short term survivor receiving the same therapeutic regimen.
  • the methods are directed to identifying or selecting a cancer patient who is expected to demonstrate enhanced survival to a therapeutic regimen comprising a proteasome inhibitor treatment regimen and/or glucocorticoid treatment regimen.
  • methods of identifying a patient who is expected to have a reduced survival time to such a therapeutic regimen e.g., as compared to a patient identified as a long term survivor on the same therapeutic regimen.
  • These methods typically include determining the level of expression of one or more predictive markers in a patient's tumor (e.g., a patient's cancer cells), comparing the level of expression to a reference expression level, and identifying or advising whether expression in the sample includes a pattern or profile of expression of a selected predictive marker or marker set which corresponds to expected long term or short term survival to a treatment regimen, e.g., a proteasome inhibitor treatment regimen and/or glucocorticoid treatment regimen.
  • a treatment regimen e.g., a proteasome inhibitor treatment regimen and/or glucocorticoid treatment regimen.
  • methods include therapeutic methods which further include the step of beginning, continuing, or commencing a therapy accordingly where a patient's predictive marker profile indicates that the patient is expected to demonstrate enhanced survival time with the therapy, e.g., the proteasome inhibition and/or glucocorticoid therapeutic regimen.
  • the methods include therapeutic methods which further include the step of stopping, discontinuing, altering or halting a therapy accordingly where a patient's predictive marker profile indicates that the patient is a long term survivor but is expected to demonstrate similar survival times with an alternative treatment than the proteasome inhibition and/or glucocorticoid therapeutic regimen.
  • the methods include therapeutic methods which further include the step of stopping, discontinuing, altering or halting a therapy regimen accordingly where a patient's predictive marker profile indicates that the patient is expected to demonstrate reduced survival time with the proteasome inhibition and/or glucocorticoid therapeutic regimen, e.g., as compared to a patient identified as a long term survivor receiving the same therapeutic regimen.
  • methods are provided for analysis of a patient not yet being treated with a proteasome inhibition therapy or glucocorticoid therapy and identification and prediction that the patient is expected to be a short term survivor based upon the patient's marker profile.
  • Such methods can include not being treated with the proteasome inhibition therapy and/or glucocorticoid therapy, being treated with proteosome inhibition therapy and/or glucocorticoid therapy in combination with one more additional therapies, being treated with an alternative therapy to proteosome inhibition therapy and/or glucocorticoid therapy, or being treated with a more aggressive dosing and/or administration regimen of a proteosome inhibitor and/or glucocorticoid, e.g., as compared to the dosing and/or administration regimen of a patient identified as a long term survivor.
  • the provided methods of the invention can eliminate ineffective or inappropriate use of proteasome inhibition therapy and/or glucocorticoid therapy regimens.
  • classifiers which can be used to develop a diagnostic test or a readable array useful for identifying patients who are expected to be long term or short term survivors to proteasome inhibition therapy regimen and/or glucocorticoid therapy regimen.
  • Probes or peptides identified in a classifier of the invention can be included in a diagnostic or prognostic test: to select a therapy, e.g., a proteasome inhibition therapy regimen and/or glucocorticoid therapy regimen; to determine continuation or discontinuation of therapy, e.g., a proteasome inhibition therapy regimen and/or glucocorticoid therapy regimen; or to determine a therapy regimen, e.g., a proteosome inhibition therapy regimen and/or glucocorticoid treatment regimen, should be altered, e.g., to a more aggressive therapy and/or therapy regimen.
  • Additional methods include methods to determine the activity of an agent, the efficacy of an agent, or identify new therapeutic agents or combinations. Such methods include methods to identify an agent as useful, e.g., as a proteasome inhibitor and/or a glucocorticoid inhibitor, for treating a cancer, e.g. a hematological cancer (e.g., multiple myeloma, leukemias, lymphoma, etc) or cancer from a solid tumor (e.g., in lung, breast, prostate, ovary, colon, kidney or liver), based on its ability to affect the expression of markers in a marker set of the invention.
  • a cancer e.g. a hematological cancer (e.g., multiple myeloma, leukemias, lymphoma, etc) or cancer from a solid tumor (e.g., in lung, breast, prostate, ovary, colon, kidney or liver), based on its ability to affect the expression of markers in a marker set of the invention.
  • an inhibitor which decreases or increases the level of expression of a marker or markers provided as upregulated or downregulated, respectively, in a set predictive for survival time of the patient having cancer would be a candidate inhibitor for the cancer.
  • an inhibitor which decreases or increases the level of expression of a marker or markers provided as upregulated or downregulated, respectively, in a set predictive for responsiveness to glucocorticoid inhibition of the cancer would be a candidate inhibitor for the cancer.
  • an inhibitor which decreases or increases the level of expression of a marker or markers provided as upregulated or downregulated, respectively, in a set predictive of short term or long term survival of the cancer would be an alternative candidate to proteosome inhibition and/or glucocorticoid inhibition for the cancer.
  • the present invention is also directed to methods of treating a cancer patient, with a therapeutic regimen, in particular a proteasome inhibitor therapy regimen (e.g., a proteasome inhibitor agent, alone, or in combination with an additional agent such as a chemotherapeutic agent) and/or glucocorticoid therapy regimen (a glucocorticoid agent, alone or in combination with an additional agent), which includes the step of selecting a patient whose predictive marker profile indicates that the patient is expected to be a long term survivor with the therapeutic regimen, and treating the patient with the proteasome inhibition therapy and/or glucocorticoid therapy.
  • a proteasome inhibitor therapy regimen e.g., a proteasome inhibitor agent, alone, or in combination with an additional agent
  • glucocorticoid therapy regimen a glucocorticoid agent, alone or in combination with an additional agent
  • the method can include the step of selecting a patient whose predictive marker profile indicates that the patient is expected to be a long term survivor and administering a therapy other than proteosome inhibition therapy and/or glucocorticoid therapy that demonstrates similar expected survival times as the proteosome inhibition and/or glucocorticoid therapy.
  • Additional methods of treating a cancer patient include selecting patients that are unlikely to experience enhanced survival time upon treatment with a cancer therapy (e.g., proteasome inhibition therapy, glucocorticoid therapy). Such methods can further include one or more of: administering a higher dose or increased dosing schedule of a proteosome inhibitor and/or glucocorticoid as compared to the dose or dosing schedule of a patient identified as a long term survivor; administering a cancer therapy other than proteosome inhibition therapy and/or glucocorticoid therapy; administering a proteosome inhibitor agent and/or glucocorticoid agent in combination with an additional agent. Further provided are methods for selection of a patient having aggressive disease which is expected to demonstrate more rapid time to progression and death.
  • Additional methods include a method to evaluate whether to treat or pay for the treatment of cancer, e.g. hematological cancer (e.g., multiple myeloma, leukemias, lymphoma, etc) or cancer from a solid tumor (e.g., in lung, breast, prostate, ovary, colon, kidney or liver), by reviewing a patient's predictive marker profile for long term or short term survivors to a cancer therapy, e.g., proteasome inhibition and/or glucococorticoid therapy regimen, and making a decision or advising on whether payment should be made.
  • hematological cancer e.g., multiple myeloma, leukemias, lymphoma, etc
  • cancer from a solid tumor e.g., in lung, breast, prostate, ovary, colon, kidney or liver
  • a cancer therapy e.g., proteasome inhibition and/or glucococorticoid therapy regimen
  • Figure 1 depicts bone marrow aspirate enrichment procedure effectively depletes non-tumor cells.
  • A Bone marrow aspirate samples before and after enrichment were subject to CD138 staining and FACS analysis.
  • B Myeloma purity score is elevated in control plasma cell samples (>90% pure) relative to bone marrow mononuclear cells (MNC), neutrophils, &erythroid cells.
  • MNC bone marrow mononuclear cells
  • neutrophils neutrophils
  • &erythroid cells Two enriched patient samples of 84% and 91% tumor purity by FACS analysis had scores of 35 and 28 respectively (blue arrows).
  • a score of >10 at least 3 fold elevated relative to the score non-PC cell types was set as a threshold for further analysis.
  • Figure 2 provides analysis of characteristics of the patients, samples and genes followed in the survival study.
  • Figure 2A is a table representing sample relationships which are influenced by clinical and gene-expression characteristics.
  • 264 myeloma patient samples and 6 normal plasma cell control (PC) samples were subject to unsupervised hierarchical clustering based upon 9174 differentially expressed probesets.
  • Highly related branches (labeled Groups 1-5) were identified by setting a fixed similarity metric (GeneMaths software) and requiring at least 12 samples for membership; unlabelled samples are comprised of various smaller groups.
  • Patient attributes are encoded below the sample dendrogram. Attributes with non-random distribution (p ⁇ 0.05) are marked by astericks (*). The black and white color code is described in the table.
  • Figure 2B an overview of the 100 probesets associated with survival (from Table 2), with an expansion of specific functional groups.
  • Figure 3 provides prediction of survival using Super PC.
  • an element means at least one element and can include more than one element.
  • a "marker” is a naturally-occurring polymer corresponding to at least one of the nucleic acids or proteins associated with AFFYMETRIX® probe set identifiers listed in any one of Table 1 and Table 2.
  • markers include, without limitation, sequences recognized by the Affymetric probes and probeset identifiers, sense and anti-sense strands of genomic DNA (Le. including any introns occurring therein), RNA generated by transcription of genomic DNA (Le. prior to splicing), RNA generated by splicing of RNA transcribed from genomic DNA, and proteins generated by translation of spliced RNA (Le. including proteins both before and after cleavage of normally cleaved regions such as transmembrane signal sequences).
  • a “marker” may also include a cDNA made by reverse transcription of an RNA generated by transcription of genomic DNA (including spliced RNA).
  • a “marker set” is a group of markers, comprising two or more (e.g., 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 75, 100, 200, 300 or 400) predictive markers of the invention. Markers of the present invention include the predictive markers identified in Table 1 and Table 2; as identified by the particular probeset identifier, representative public identifier, title, gene symbol, and/or Entrez gene identifier, and include the representative nucleotide and/or protein sequence or fragment thereof which corresponds to the identifier.
  • a "predictive marker” as used herein includes a marker which has been identified as having differential expression in tumor cells of a patient and furthermore that expression is characteristic of a patient whose survival time is expected to be longer or shorter with treatment of a proteasome inhibitor and/or glucocorticoid.
  • a predictive marker includes a marker which demonstrates higher expression in a short term survival patient; alternatively a predictive marker includes a marker which demonstrates higher expression in a long term survival patient.
  • a predictive marker is intended to include those markers which demonstrate lower expression in a short term survival patient as well as those markers which demonstrate lower expression in a long term survival patient.
  • predictive marker is intended to include each and every one of these possibilities, and further can include each single marker individually as a predictive marker; or alternatively can include one or more, or all of the characteristics collectively when reference is made to "predictive markers” or “predictive marker sets.”
  • a predictive marker set also can be known as a "classifier.”
  • a "naturally-occurring” refers to a molecule (e.g., RNA, DNA, protein, etc.) that occurs in nature (e.g., encodes a natural protein, a naturally produced protein, etc).
  • probe refers to any molecule which is capable of selectively binding to a specifically intended target molecule, for example a marker of the invention. Probes can be either synthesized by one skilled in the art, or derived from appropriate biological preparations. For purposes of detection of the target molecule, probes may be specifically designed to be labeled, as described herein. Examples of molecules that can be utilized as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic monomers.
  • the "normal" level of expression of a marker is the level of expression of the marker in cells in a similar environment or response situation, in a patient not afflicted with cancer.
  • a normal level of expression of a marker may also refer to the level of expression of a "reference sample", (e.g., sample from a healthy subjects not having the marker associated disease).
  • a reference sample expression may be comprised of an expression level of one or more markers from a reference database.
  • a "normal" level of expression of a marker is the level of expression of the marker in non-tumor cells in a similar environment or response situation from the same patient that the tumor is derived from.
  • “Differential expression” of a marker refers to expression of a marker that varies in level across patients. Furthermore, in this invention we refer to a marker as "differentially expressed” when its expression level is correlated with, or otherwise indicative of, long term or short term survival associated with a treatment.
  • “Complementary” refers to the broad concept of sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that an adenine residue of a first nucleic acid region is capable of forming specific hydrogen bonds ("base pairing") with a residue of a second nucleic acid region which is antiparallel to the first region if the residue is thymine or uracil. Similarly, it is known that a cytosine residue of a first nucleic acid strand is capable of base pairing with a residue of a second nucleic acid strand which is antiparallel to the first strand if the residue is guanine.
  • a first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if, when the two regions are arranged in an antiparallel fashion, at least one nucleotide residue of the first region is capable of base pairing with a residue of the second region.
  • the first region comprises a first portion and the second region comprises a second portion, whereby, when the first and second portions are arranged in an antiparallel fashion, at least about 50%, and preferably at least about 75%, at least about 90%, or at least about 95% of the nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion. More preferably, all nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion.
  • informative expression is intended to refer to the expression level of a differentially expressed predictive marker which corresponds to short term or long term survival.
  • the expression level of a marker in a patient is “informative” if it is greater than a reference level by an amount greater than the standard error of the assay employed to assess expression.
  • the informative expression level of a marker can be determined upon statistical correlation of the measured expression level and the outcome, e.g. short term or long term survival. The result of the statistical analysis can establish a threshold for selecting markers to use in the methods described herein.
  • a marker that is differentially expressed will have typical ranges of expression level that are predictive of short term and long term survival.
  • An informative expression level is a level that falls within the short term and long term survival range of expressions. Still further, a set of markers may together be "informative" if the combination of their expression levels either meets or is above or below a pre-determined score for a predictive marker set as determined by methods provided herein.
  • a given marker may be indicative of both short term and long term survival in patients; for example, expression of a predictive marker provided herein above a given threshold (e.g., an informative expression level) may be indicative of long term survival in a patient, as described herein. Expression of that marker below a given threshold (e.g., below an informative level) may be indicative of short term survival in a patient
  • a cancer or tumor is treated or diagnosed according to the present methods.
  • Cancer or “tumor” is intended to include any neoplastic growth in a patient, including an inititial tumor and any metastases.
  • the cancer can be of the liquid or solid tumor type.
  • Liquid tumors include tumors of hematological origin, including, e.g., myelomas (e.g., multiple myeloma), leukemias (e.g., Waldenstrom's syndrome, chronic lymphocytic leukemia, other leukemias), and lymphomas (e.g., B-cell lymphomas, non-Hodgkins lymphoma).
  • Solid tumors can originate in organs, and include cancers such as lung, breast, prostate, ovary, colon, kidney, and liver.
  • cancer cells refer to cells that divide at an abnormal (increased) rate.
  • Cancer cells include, but are not limited to, carcinomas, such as squamous cell carcinoma, basal cell carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, adenocarcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, undifferentiated carcinoma, bronchogenic carcinoma, melanoma, renal cell carcinoma, hepatoma- liver cell carcinoma, bile duct carcinoma, cholangiocarcinoma, papillary carcinoma, transitional cell carcinoma, choriocarcinoma, semonoma, embryonal carcinoma, mammary carcinomas, gastrointestinal carcinoma, colonic carcinomas, bladder carcinoma, prostate carcinoma, and squamous cell carcinoma of the neck and head region; sarcomas, such as fibrosarcoma, myxosarcoma, Iiposarcoma, chondros
  • carcinomas such as
  • long term survivor and “short term survivor” refer to the length of time after receiving a first dose of treatment that a cancer patient is predicted to live.
  • a “long term survivor” refers to a patient expected have a slower rate of progression and death from the tumor than those patients identified as short term survivors.
  • Enhanced survival or “a slower rate of death” are estimated life span determinations based upon elevated or reduced expression of a sufficient number of predictive markers from Table 1 and/or Table 2 as compared to a reference standard such that 70%, 80%, 90% or more of the population will be alive a sufficient time period after receiving a first dose of treatment.
  • a “faster rate of death” or “shorter survival time” refer to estimated life span determinations based upon elevated or reduced expression of a sufficient number of predicted markers from Table 1 and/or Table 2 as compared to a reference standard such that 50%, 40%, 30%, 20%, 10% or less of the population will not live a sufficient time period after receiving a first dose of treatment.
  • the sufficient time period is at least 6, 12, 18, 24 or 30 months measured from the first day of receiving a cancer therapy.
  • Treatment shall mean the use of a therapy to prevent or inhibit further tumor growth, as well as to cause shrinkage of a tumor, and to provide longer survival times. Treatment is also intended to include prevention of metastasis of tumor.
  • a tumor is "inhibited” or “treated” if at least one symptom (as determined by responsiveness/non-responsiveness, time to progression, or indicators known in the art and described herein) of the cancer or tumor is alleviated, terminated, slowed, minimized, or prevented. Any amelioration of any symptom, physical or otherwise, of a tumor pursuant to treatment using a therapeutic regimen (e.g., proteasome inhibition regimen, glucocorticoid regimen) as further described herein, is within the scope of the invention.
  • a therapeutic regimen e.g., proteasome inhibition regimen, glucocorticoid regimen
  • agents are defined broadly as anything that cancer cells, including tumor cells, may be exposed to in a therapeutic protocol.
  • agents include, but are not limited to, proteasome inhibition agents, glucocorticoidal steroid agents, as well as chemotherapeutic agents as known in the art and described in further detail herein.
  • kits are any article of manufacture (e.g. a package or container) comprising at least one reagent, e.g. a probe, for specifically detecting a marker or marker set of the invention.
  • the article of manufacture may be promoted, distributed, sold or offered for sale as a unit for performing the methods of the present invention.
  • the reagents included in such a kit comprise probes/primers and/or antibodies for use in detecting short term and long term survival marker expression.
  • the kits of the present invention may preferably contain instructions which describe a suitable detection assay.
  • kits can be conveniently used, e.g., in clinical settings, to diagnose and evaluate patients exhibiting symptoms of cancer, in particular patients exhibiting the possible presence of an a cancer capable of treatment with proteasome inhibition therapy and/or glucocorticoid therapy, including, e.g., hematological cancers e.g., myelomas (e.g., multiple myeloma), lymphomas (e.g., non-hodgkins lymphoma), leukemias, and solid tumors (e.g., lung, breast, ovarian, etc.).
  • the present methods and compositions are designed for use in diagnostics and therapeutics for a patient suffering from cancer.
  • the cancer can be of the liquid or solid tumor type.
  • Liquid tumors include tumors of hematological origin, including, e.g., myelomas (e.g., multiple myeloma), leukemias (e.g., Waldenstrom's syndrome, chronic lymphocytic leukemia, other leukemias), and lymphomas (e.g., B-cell lymphomas, non-Hodgkins lymphoma).
  • myelomas e.g., multiple myeloma
  • leukemias e.g., Waldenstrom's syndrome, chronic lymphocytic leukemia, other leukemias
  • lymphomas e.g., B-cell lymphomas, non-Hodgkins lymphoma
  • Solid tumors can originate in organs, and include cancers such as lung, breast, prostate, ovary, colon, kidney, and liver.
  • the invention provides methods for determining, assessing, advising or providing an appropriate cancer therapy regimen for treating a tumor in a patient.
  • the cancer therapy regimens appropriate for use in or in conjunction with the provided methods include proteasome inhibition therapy regimens and/or glucocorticoid therapy regimens.
  • proteasome inhibitor therapy comprises treatment of a patient with a proteasome inhibitor (e.g., bortezomib, or any other proteasome inhibitor described in further detail herein), alone or in combination with one or more additional agents.
  • glucocorticoid therapy comprises treatment of a patient with a glucocorticoid (e.g., dexamethasone, or any other glucocorticoid described in further detail herein), alone or in combination with one or more additional agents.
  • a cancer therapy regimen also refers to dose amounts, the frequency of dosing and the number of times a cancer therapy is administered.
  • dosing schedule or “administration schedule” as used herein refer to both the frequency of dosing and the number of times a cancer therapy is administered.
  • the provided methods comprise measuring the level of expression of at least one predictive marker in the patient's tumor and determining or advising on a cancer therapy regimen for treating the tumor based on the expression level of the predictive marker or markers, as relevant.
  • An informative expression level of a predictive marker or markers in the patient sample is an indication that the patient is expected to exhibit longer survival time and would benefit from proteasome inhibition therapy and/or glucocorticoid therapy when the predictive marker or marker set provided herein indicate such survival times.
  • An informative expression level of a predictive marker or markers in the patient sample can also indicate that the patient is expected to exhibit longer survival time and would benefit from an alternative cancer therapy other than proteosome inhibition and/or glucocorticoid therapy that provides similar expectation of survival as the proteosome inhibition and/or glucocorticoid therapy.
  • an informative expression level of a predictive marker or markers in a patient is an indication that the patient is not expected to have a long survival time and would not benefit from proteasome inhibition therapy and/or glucocorticoid therapy, or may need a more aggressive therapeutic regimen (e.g., dosing and/or administration regimen) with proteosome inhibition and/or glucocorticoid therapy than a patient classified as a long term survivor when the marker or markers provided herein indicate such short term survival.
  • a more aggressive therapeutic regimen e.g., dosing and/or administration regimen
  • the invention further provides methods for determining or advising whether a patient is expected to be a long term survivor in response to a cancer therapy regimen for treating a tumor.
  • Such methods comprise measuring the level of expression of at least one predictive marker in the patient's tumor and determining, advising or providing a proteasome inhibition based regimen and/or glucocorticoid based regimen for treating the tumor based on the expression level of the predictive marker or marker set.
  • An informative expression level of a predictive marker in the patient sample is an indication that the patient is expected to demonstrate long term survival and would benefit from proteasome inhibition and/or glucocorticoid therapy.
  • An informative expression level of a predictive marker set in the patient is an indication that the patient is expected to demonstrate long term survival and would benefit from proteasome inhibition therapy and/or glucocorticoid therapy when the marker or markers provided herein indicate such long term survival.
  • An informative expression level of a predictive marker or markers in the patient sample can also indicate that the patient is expected to exhibit longer survival time and would benefit from an alternative cancer therapy other than proteosome inhibition and/or glucocorticoid therapy that provides similar expectation of survival time as the proteosome inhibition and/or glucocorticoid therapy.
  • Selected predictive markers for use in the methods comprise predictive markers which demonstrate increased expression in long term survival patients and/or which are expected to show longer time to disease progression and death and, e.g., are not specific to treatment with proteosome inhibition therapy or glucocorticoid therapy.
  • the invention provides methods for determining or advising whether a patient has aggressive disease and is predicted to progress in disease and to death faster than a patient not demonstrating aggressive disease.
  • a patient indicative of having aggressive disease also may be predicted to have short survival time in response to a cancer therapy regimen for treating a tumor.
  • Such methods comprise measuring the level of expression of at least one predictive marker in the patient's tumor and identifying the patient as having aggressive disease based on the expression level of the predictive marker or marker set.
  • An informative expression level of a predictive marker in the patient sample is an indication that the patient has aggressive disease patient and is likely to progress to death more rapidly than a patient determined to be a long term survivor and may not benefit from proteasome inhibition based regimen and/or glucocorticoid based regimen therapy, or may need a more aggressive therapy regimen (e.g., dosing and/or administration regimen) with proteosome inhibition and/or glucocorticoid therapy than a patient classified as a long term survivor.
  • a more aggressive therapy regimen e.g., dosing and/or administration regimen
  • An informative expression level of a predictive marker set in the patient is an indication that the patient is a patient having aggressive disease and would not benefit from proteasome inhibition based regimen and/or glucocorticoid based regimen, or may need a more aggressive therapeutic regimen (e.g., dose and/or administration schedule) with proteosome inhibition and/or glucocorticoid therapy than a patient classified as a long term survivor when the selected marker or marker set provided herein indicate such disease aggressiveness.
  • Selected predictive markers for use in the methods comprise predictive markers which demonstrate increased expression in short term survival patients and/or shorter time to disease progression and death in patients and are not specific to treatment with proteasome inhibition therapy or glucocorticoid therapy.
  • the method can further include determining, advising or providing: an alternative cancer therapy than proteosome inhibition therapy and/or glucocorticoid therapy; an additional cancer therapy or therapies in conjunction with the proteosome inhibition therapy and/or glucocorticoid therapy; alternative dose and/or administration schedule, e.g., than determined, advised or provided for a patient predicted to be a long term survival patient, of a proteosome inhibition therapy and/or glucocorticoid therapy.
  • the invention further provides methods for treating a tumor in a patient with a proteasome inhibition based therapy regimen and/or glucocorticoid based therapy regimen.
  • Such therapeutic methods comprise measuring the level of expression of at least one predictive marker in a patient's tumor; determining or advising whether a proteasome inhibition based regimen and/or glucocorticoid based regimen for treating the tumor is appropriate based on the expression level of the predictive marker or markers, and treating a patient with a proteasome inhibition based therapy and/or glucocorticoid based therapy when the patient's expression level indicates a a long term survival patient.
  • An informative expression level of predictive marker in the patient sample is an indication that the patient is a long term survival patient and would benefit from proteasome inhibition based regimen and/or glucocorticoid based regimen therapy when the predictive marker or marker set provided herein indicate the patient is a long term survival patient.
  • the invention further provides methods for treating a tumor in a patient with a cancer therapy other than a proteosome inhibition based regimen and/or glucocorticoid based regimen that is predicted to have demonstrate similar survival times.
  • Such therapeutic methods comprise measuring the level of expression of at least one predictive marker in a patient's tumor; determining or advising whether a proteasome inhibition based regimen and/or glucocorticoid based regimen for treating the tumor is appropriate based on the expression level of the predictive marker or markers, and treating a patient with the alternative cancer therapy when the patient's expression level indicates a long term survival patient.
  • An informative expression level of predictive marker in the patient sample is an indication that the patient is a long term survival patient and would benefit from the alternative cancer therapy when the predictive marker or marker set provided herein indicate the patient is a long term survival patient.
  • the invention provides methods for treating a tumor in a patient identified as a short term survival patient.
  • Such therapeutic methods comprise determining or advising on a cancer therapy regimen based upon expression of at least one predictive marker in a patient's tumor, and treating a patient with the cancer therapy regimen when the patient's expression level indicates a short term survival patient.
  • a cancer therapy regimen can be: a cancer therapy regimen other than a proteosome inhibition therapy regimen and/or glucocorticoid therapy regimen; an additional cancer therapy or therapies administered in conjuction with the proteosome inhibition therapy and/or glucocorticoid therapy; alternative dosing and/or dosage administration, e.g., than determined, advised or provided for a patient predicted to be a long term survival patient, of a proteosome inhibition therapy and/or glucocorticoid therapy.
  • Methods of the invention use at least one of the predictive markers set forth in any one of Table 1 and Table 2. Additionally, the methods provided can use two, three, four, five, six, or more markers to form a predictive marker set.
  • marker sets selected from the markers in Table 1 and Table 2 can be generated using the methods provided herein and can comprise between two, and all of the markers set forth in Table land/or Table 2 and each and every combination in between (e.g., four selected markers, 16 selected markers, 74 selected markers, etc.).
  • the predictive marker set comprises at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 100, 150, 200, or 300 or more markers.
  • the predictive marker set comprises no more than 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 100, 150, 200, 300, 400, 500, 600, 700, 1,000, 2,000 markers.
  • the predictive marker set includes a plurality of genes associated with cancer, e.g. a hematological cancer (e.g., multiple myeloma, leukemias, lymphoma, etc) or cancer from a solid tumor (e.g., in lung, breast, prostate, ovary, colon, kidney or liver).
  • the predictive marker set includes a plurality of markers listed in Table 1 and Table 2.
  • the predictive marker set includes at least about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% of the markers listed in Table 1 and/or Table 2.
  • Selected predictive marker sets can be assembled from the predictive markers provided using methods provided herein and analogous methods known in the art.
  • Methods of the invention further provide the ability to construct marker sets from the individual predictive markers set forth in Table 1, and Table 2 using the methods described in further detail herein.
  • more than one marker set can be used in combination for the diagnostic, prognostic and treatment methods provided.
  • the methods of the invention can be performed such that determination of the level of expression of a predictive marker is measured prior to tumor therapy in order to identify whether the patient is predicted to demonstrate long term survival with a particular cancer therapy regimen, e.g., a proteasome inhibition therapy regimen and/or glucocorticoid therapy regimen.
  • the methods of the invention can be performed concurrently with ongoing tumor therapy to determine if long term survival is predicted for a patient receiving proteasome inhibition therapy and/or glucocorticoid therapy or predicted for a patient who will receive additional therapy comprising proteasome inhibition therapy and/or glucocorticoid therapy.
  • the methods of the invention can be performed after a tumor therapy has been carried out in order to assess whether the patient is predicted to demonstrate long term survival and/or whether additional cancer therapy regimens should be carried out. Such methods can also be performed to assess future cancer therapy regimens, e.g., future proteosome inhibition therapy regimens and/or glucocorticoid therapy regimens, for the patient.
  • the tumor therapy can comprise proteasome inhibition therapy and/or glucocorticoid therapy, alone or alternative forms of cancer therapy.
  • the methods can determine if the patient will benefit from additional or future proteasome inhibition and/or glucocorticoid therapy regimens, and can include such proteasome inhibition and/or glucocorticoid therapy alone or in combination with additional therapeutic agents.
  • the level of expression of predictive marker in the patient's tumor is measured by isolating a sample of the tumor and performing analysis on the isolated sample, or a portion thereof. In another aspect, the level of expression of predictive marker in the patient's tumor is measured using in vivo imaging techniques.
  • determining the level of expression of a predictive marker comprises detection of mRNA. Such detection can be carried out by any relevant method, • including e.g., PCR, northern, nucleotide array detection, in vivo imaging using probes capable of detection of the appropriate nucleic acid. In other aspects, determining the level of expression of the predictive marker comprises detection of protein. Such detection can be carried out using any relevant method for protein detection, including e.g., ELISA, western blot, immunoassay, protein array detection, in vivo imaging using probes capable of detection of the appropriate peptide. [0053] Determining the level of expression of a predictive marker is compared to a reference expression level.
  • a reference ' expression level can be a predetermined standard reference level of expression in order to evaluate if expression of a marker or marker set is informative and make an assessment for determining whether the patient is a short term or long term survivor.
  • determining the level of expression of a predictive marker can be compared to an internal reference marker level of expression which is measured at the same time as the predictive marker in order to make an assessment for determining whether the patient is a short term or long term survivor.
  • expression of a distinct marker or markers which is/are not predictive markers of the invention, but which is known to demonstrate a constant expression level can be assessed as an internal reference marker level, and the level of the predictive marker expression is determined as compared to the reference.
  • expression of the selected predictive marker or markers in a tissue sample which is a non-tumor sample can be assessed as an internal reference marker level.
  • the level of expression of a marker or markers may be determined as having increased expression in certain aspects.
  • the level of expression of a marker or markers may be determined as having decreased expression in other aspects.
  • the level of expression may be determined as no informative change in expression as compared to a reference level.
  • the level of expression is determined against a pre-determined standard expression level as determined by the methods provided herein.
  • the invention also relates to various reagents and kits for diagnosing, staging, prognosing, monitoring and treating a cancer patient (e.g., a patient with a liquid tumor or a solid tumor), with a cancer therapy regimen, e.g., proteasome inhibition therapy and/or glucocorticoid therapy regimens.
  • a cancer patient e.g., a patient with a liquid tumor or a solid tumor
  • a cancer therapy regimen e.g., proteasome inhibition therapy and/or glucocorticoid therapy regimens.
  • reagents for detection of markers and marker sets and for use in the methods of the invention comprising at least two isolated predictive markers set forth in Table 1 and Table 2.
  • Such reagents include nucleic acid probes, primers, antibodies, antibody derivatives, antibody fragments, and peptide probes for detection of the relevant predictive markers set forth in Table 1 and Table 2.
  • kits for use in the provided methods include reagents for assessing predictive markers (e.g., at least one predictive marker) and predictive marker sets (e.g., at least two, three, four or more markers selected from Table 1 and Table 2), as well as instructions for use in accordance with the methods provided herein.
  • the kits provided contain nucleic acid probes for assessment of predictive markers.
  • the kits provided contain antibody, antibody derivative antibody fragment, or peptide reagents for assessment of predictive markers.
  • the present invention provides markers that are expressed in a tumor that predict enhanced survival times in a patient receiving a cancer therapy, e.g., proteasome inhibition therapy and/or glucocorticoid therapy, and whose expression correlates with longer survival times in such patients.
  • the present invention also provides markers that are expressed in a tumor that predict shorter survival times for patients receiving a cancer therapy, e.g., a proteasome inhibition therapy and/or glucocorticoid therapy, and whose expression correlates with shorter survival times in such patients. Accordingly, one or more of the markers can be used to identify cancers that can be successfully treated by proteasome inhibition therapy regimens and/or glucocorticoid therapy regimens.
  • markers of the present invention can be used to identify patients that can be successfully treated using proteasome inhibition therapy regimens and/or glucocorticoid therapy regimens.
  • the markers of the present invention can be used to identify a patient that has become or is at risk of becoming refractory to treatment with proteasome inhibition therapy and/or glucocorticoid therapy.
  • the invention also features combinations of markers, referred to herein as "marker sets,” that can predict whether a patient is likely to demonstrate long term or short term survival to a cancer therapy regimen, e.g., proteasome inhibition therapy and/or glucocorticoid therapy regimens.
  • Table 1 sets forth predictive markers identified using statistical analysis applied to samples from 264 patients, which are specific identifiers of overall survival times (OS) in patients receiving proteasome inhibition therapy (e.g., bortezomib) or glucocorticoid therapy (e.g., dexamethasone).
  • OS overall survival times
  • glucocorticoid therapy e.g., dexamethasone
  • the markers in Table 1 are correlated with a predicted time until death as determined by a Cox proportional hazard analysis, as described in further detail herein.
  • Table 2 also sets forth predictive markers identified using statistical analysis but was derived from a subset of the patients evaluated for the data in Table 1 and was determined using the superpc method of Bair and Tibshirani, as described in further detail herein.
  • the predictive markers of Table 2 are also specific identifiers of overall survival times (OS) in patients receiving proteasome inhibition therapy (e.g., bortezomib) or glucocorticoid therapy (e.g., dexamethasone).
  • the markers in Table 1 and Table 2 are differentially expressed in samples from patients that are predicted to demonstrate short term (“short term survivor) or long term survival ("long term survivor") with the proteosome inhibitor bortezomib or the glucocorticoid dexamethasone.
  • the markers identified can function in a predictive model to prospectively identify patients expected to survive for longer periods when treated with proteosome inhibition therapy, including bortezomib or other proteasome inhibition therapies known in the art as well as those described in further detail herein, and/or glucocorticoid therapy, including dexamethasone or other glucocorticoids known in the art as well as those described in further detail herein.
  • proteosome inhibition therapy including bortezomib or other proteasome inhibition therapies known in the art as well as those described in further detail herein
  • glucocorticoid therapy including dexamethasone or other glucocorticoids known in the art as well as those described in further detail herein.
  • Predictors of long time to death are useful as indicators of patients who are likely to progress to death at a slower rate and may be more likely to be responsive to therapy than other patients.
  • the predictive markers in Table 1 and Table 2 are correlated with a predicted short time to death ("short term survivors").
  • Table 1 and Table 2 provide predictive markers which are upregulated indicators correlated with shorter time to death. Table 1 and Table 2 also provide predictive markers which are upregulated indicators correlated with longer time to death. Table 1 indicates whether a marker also is identified as a marker for responsiveness or non-responsiveness to a treatment (proteasome inhibition therapy or dexamethasone therapy; see, International Patent Publication No. WO04053066, published June 24, 2004, or U.S. Patent Application No. 11/449,195, filed June 8, 2006, the entire contents of each application incorporated herein by reference). [0059] In the methods of the present invention, the level of expression of one or more predictive markers selected from the group consisting of the markers identified in Table 1 and Table 2 is determined. As used herein, the level or amount of expression refers to the absolute level of expression of an mRNA encoded by the marker or the absolute level of expression of the protein encoded by the marker (i.e., whether or not expression is or is not occurring in the cancer cells).
  • Marker sets comprising the predictive markers identified herein can be generated using the methods and predictive markers provided. Thus, it is possible to assess the expression of a panel of short term and long term survival markers using the methods and compositions provided herein.
  • determinations may be based on normalized expression levels.
  • Expression levels are normalized by correcting the absolute expression level of a predictive marker by comparing its expression to the expression of a reference marker that is not a predictive marker, e.g., a housekeeping gene that is constitutively expressed.
  • Suitable markers for normalization include housekeeping genes, such as the actin gene.
  • Constitutively expressed genes are known in the art and can be identified and selected according to the relevant tissue and/or situation of the patient and the analysis methods. Such normalization allows one to compare the expression level in one sample, e.g., a tumor sample, to another sample, e.g., a non-tumor sample, or between samples from different sources.
  • the expression level can be provided as a relative expression level.
  • the level of expression of the predictive marker or marker set is determined for 10 or more individual samples, preferably 50 or more individual samples in order to establish a baseline, prior to the determination of the expression level for the sample in question.
  • mean expression level of each of the predictive markers or marker sets assayed in the larger number of samples is determined and this is used as a baseline expression level for the predictive markers or marker sets in question.
  • the expression level of the marker or marker set determined for the test sample is then divided by the mean expression value obtained for that marker or marker set. This provides a relative expression level and aids in identifying extreme cases of short term or long term survival times. Determining Short Term and Long Term Survival
  • the expression level (including protein level) of the identified predictive markers of short term/long term survival patients may be used to: 1) determine if a patient can be treated by an agent or combination of agents; 2) determine if a patient is responding to treatment with an agent or combination of agents; 3) select an appropriate agent or combination of agents for treating a patient; 4) select an appropriate dosing and/or administration schedule of an agent or agents; 5) monitor the effectiveness of an ongoing treatment; 6) identify new cancer therapy treatments (either single agent proteasome inhibitor and/or glucocorticoid agents or complementary agents which can be used alternatively or in combination with proteasome inhibition and/or glucocorticoid agents); and 7) identify aggressiveness of a cancer.
  • the identified predictive markers may be utilized to determine appropriate therapy, to monitor clinical therapy and human trials of a drug being tested for efficacy, and to develop new agents and therapeutic combinations.
  • a patient being treated with an agent may exhibit a longer time to death if one or more of the corresponding predictive markers identified in rows 225 to 403 Table 1 and/or rows 38 to 100 of Table 2 demonstrate increased expression.
  • predisposition of a patient being treated with an agent to exhibit a longer time to death is determined by the methods of the present invention, wherein a marker set can be generated using to the methods described herein and include a subset of the markers identified in rows 225 to 403 of Table 1 and/or rows 38 to 100 of Table 2, and the expression of the marker set is evaluated.
  • a patient may exhibit a shorter time to death if one or more of the corresponding predictive markers demonstrates informative expression levels.
  • a patient may exhibit a shorter time to death if one or more of the corresponding predictive markers identified in rows 1 to 224 of Table 1 and rows 1 to 37 of Table 2 demonstrate informative increased expression.
  • predisposition of a patient being treated with an agent to exhibit a shorter time to death is determined by the methods of the present invention, wherein a marker set can be generated using to the methods described herein and include a subset of the markers identified in rows 1 to 224 of Table 1 and/or rows 1 to 37 of Table 2, and the expression of the marker set is evaluated.
  • a method of the invention can include determining the expression level of one or more markers, e.g., a plurality of markers, (e.g., 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 75, 80, 90, 100, 125, 150, or 200 markers) from Table 1 whose hazard ratio is above a particular threshold, e.g. 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2 or 3.5, preferably above 1.5, 2.0, 2.5 or 3.0.
  • a particular threshold e.g. 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2 or 3.5, preferably above 1.5, 2.0, 2.5 or 3.0.
  • a score compiled from expression levels of the markers predicts short term survival if the expression of a certain percentage of the markers, e.g., 50%, 60%, 70%, 80%, 90% or 95% of the markers show high expression.
  • a method of the invention can include determining the expression level of one or more markers, e.g.
  • markers e.g., 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 75, 80, 90, 100, 125, 150, or 175 markers
  • Table 1 whose hazard ratio is below a particular threshold, e.g., 0.90, 0.80, 0.70, 0.60, 0.50, 0.40, or 0.30, preferably below 0.75, 0.65, 0.55, 0.45 or 0.35.
  • a score compiled from expression levels of the markers predicts long term survival if the expression of a certain percentage of the markers, e.g., 50%, 60%, 70%, 80%, 90% or 95% of the markers show high expression.
  • a method of the invention can include determining the expression level of a combination of markers, some, e.g., a plurality of markers, (e.g., 10, 20, 30, 40, 50, 60) of whose hazard ratio is above a certain level, e.g., 3.0, 2.5, 2.0 or 1.5 and others, e.g., a plurality of markers, (e.g., 10, 20, 30, 40, 50, 60) of whose hazard ratio is below a certain level, e.g., 0.75, 0.65, 0.55 or 0.45 to develop a score wherein high expression level of a higher percentage of markers with high hazard ratios predicts short term survival and high expression level of a higher percentage of markers with low hazard ratios predicts long term survival.
  • a certain level e.g., 3.0, 2.5, 2.0 or 1.5
  • others e.g., a plurality of markers, (e.g., 10, 20, 30, 40, 50, 60) of whose hazard ratio is below
  • An exemplary method can measure the expression levels of 10, 15, 20 or 25 markers from Table 1 with hazard ratios at least 3.0 or 2.8 and the expression levels of 10, 15, 20 or 25 markers from Table 1 with hazard ratios no higher than 0.40 or 0.45 and further combine the levels of expression of such a combination of markers into a score from which short term survival or long term survival can be predicted by the relative percentage or weight of short term survival or long term survival markers having high expression levels.
  • a method of the invention can include determining the expression level of one or more markers, e.g. a plurality of markers, (e.g., 5, 10, 15, 20, 25, 30, or 35 markers) from Table 2 whose SuperPC score is above a particular threshold, e.g. 2.2, 2.4, 2.6, 2.8, or 3.0, preferably above 2.3, 2.5, 2.7 or 2.9.
  • a score compiled from expression levels of the markers predicts short term survival if the expression of a certain percentage of the markers, e.g., 50%, 60%, 70%, 80%, 90% or 95% of the markers show high expression.
  • a method of the invention can include determining the expression level of one or more markers, e.g.
  • markers e.g., 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or 55, markers
  • Table 2 whose SuperPC score is below a particular threshold, e.g., -2.2, -2.4, -2.6, -2.8, or -3.0, preferably below -2.3, -2.5, -2.7 or -2.9.
  • a score compiled from expression levels of the markers predicts long term survival if the expression of a certain percentage of the markers, e.g., 50%, 60%, 70%, 80%, 90% or 95% of the markers show high expression.
  • a method of the invention can include determining the expression level of one or more markers, e.g. a plurality of markers, (e.g., 5, 10, 15, 20, 25, 30, or 35 markers) from Table 2 whose absolute value of the SuperPC score is above a particular threshold, e.g. 2.2, 2.4, 2.6, 2.8, or 3.0, preferably above 2.3, 2.5, 2.7 or 2.9.
  • a particular threshold e.g. 2.2, 2.4, 2.6, 2.8, or 3.0, preferably above 2.3, 2.5, 2.7 or 2.9.
  • an exemplary method measuring the expression level of at least 5, 10, or 15 markers with an absolute score of at least 2.8 can determine the level of expression of one marker with a SuperPC score of at least 3.0, one or two markers with a SuperPC score of no higher than -3.0, one, two or three markers with a SuperPC score of at least 2.90, one two or three markers with a SuperPC score of no higher than -2.90, one, two, three or four markers with a SuperPC score of at least 2.80 and/or one, two or three markers with a SuperPC score no higher than -2.80.
  • Table G below can guide the selection of thresholds to identify markers from Table 2 to include in such a method to identify proteasome inhibition therapy or glucacorticoid inhibition therapy.
  • a method determining the expression levels of markers SuperPC score has an absolute value threshold of 2.9 (using about 8 markers from Table 2), 2.7 (using about 23 markers from Table 2), 2.5 or 2.4 (using about 53 or about 72 markers from Table 2, respectively) or 2.3 (using about 95 markers from Table 2) can predict survival outcome ofproteasome inhibition, e.g., bortezomib therapy.
  • a method determining the expression levels of markers whose SuperPC scores have an absolute value threshold of 2.9 using about 8 markers from Table 2), 2.8 (using about 16 markers from Table 2), or 2.6 (using about 37 markers from Table 2) can predict survival outcome .of glucocorticoid, e.g., dexamethasone therapy.
  • the method can include determining the expression level of markers associated with particular biological pathways or categories.
  • Tables 1 and 2 identify markers which have been annotated to particular categories or pathways to provide guidance in selecting markers to test. Markers can be selected at least from the categories of oncogenes, tumor suppressor pathway, cancer antigens, NF-KB pathway, hematopoiesis, apoptotic signaling, mitotic signaling, protein homeostasis, oncogenic signaling, adhesion, cell cycle, ubiquitin/proteasome pathway, stem cell, mitochondria function, rapamycin regulated, expressed in lymphoma, expressed in breast cancer, expressed in renal cancer, and/or RNA processing.
  • a method of the invention can include determining the level of expression of markers involved in ubiquitin or proteasome pathway, e.g., markers corresponding proteasome subunits, mitochondrial function, e.g., markers corresponding to mitochondrial ribosome proteins, cancer antigens, e.g.,markers corresponding to synovial sarcoma, X breakpoint proteins and/or stem cell markers to predict short term survival.
  • markers involved in ubiquitin or proteasome pathway e.g., markers corresponding proteasome subunits
  • mitochondrial function e.g., markers corresponding to mitochondrial ribosome proteins
  • cancer antigens e.g.,markers corresponding to synovial sarcoma
  • X breakpoint proteins e.g., X breakpoint proteins
  • a method of the invention can include determining the level of expression of markers involved in hematopoiesis, e.g., glycophorin A or B, ankyrin 1, CD36, or myosin light polypeptide 4, and/or adhesion, e.g., tenascin XB, or catenin to predict long term survival. Additional markers can be selected from these categories and are included in Tables 1 or 2 or are readily available to those skilled in the art. Methods of the invention can include a combination of measuring markers from specific categories and measuring markers beyond certain thresholds, as described in the preceding paragraphs. Reagents for measuring the protein or nucleic acid levels of markers annotated according to biological categories are readily obtained from the public knowledge of the respective sequences or are commercially available, as described in later sections.
  • the predictive marker set for evaluation of expected survival time in a patient having cancer comprises markers selected from those set forth in any of Table 1 and Table 2. Still a further aspect contemplates markers set forth in either Table 1 alone or in combination with markers set for the in Table 2, or alternatively, those markers set forth in Table 2 alone or in combination with Table 1.
  • a marker set can include all the markers set forth in Table 2.
  • a marker set can include all the markers set forth in Table 1.
  • proteasome inhibition therapy and/or glucocorticoid therapy could be continued where the expression profile indicates long term survival using the evaluation methods described herein.
  • protesome inhibition therapy and/or glucocorticoid therapy could be continued but at a more aggressive dose and/or administration schedule where the expression profile indicates short term survival using the evaluation methods described herein.
  • the present invention provides methods for determining whether a cancer therapy e.g., a proteasome inhibitor and/or glucocorticoid agent, can be used which increases the likelihood that a patient will have a slower time to death comprising evaluating expression of at least one predictive marker or a predictive marker set in a tumor sample; and identifying and/or advising that proteasome inhibition therapy and/or glucocorticoid therapy is or is not appropriate or that a dosing or administration schedule is appropriate or is not appropriate to increase the likelihood that a patient will have a slower time to death based on the evaluation.
  • a cancer therapy e.g., a proteasome inhibitor and/or glucocorticoid agent
  • the invention provides a method for determining whether a proteasome inhibition therapeutic regimen (e.g., a proteasome inhibitor agent (e.g., bortezomib) alone or in combination with another chemotherapeutic agent) to increase the likelihood that a patient will have a slower time to death comprising determining the expression profile of a predictive marker or predictive marker set; and identifying and/or advising that a proteasome inhibition therapeutic agent is or is not appropriate or that a dosing or administration schedule is appropriate or is not appropriate to increase the likelihood that a patient will have a slower time to death based on the expression profile.
  • a proteasome inhibition therapeutic regimen e.g., a proteasome inhibitor agent (e.g., bortezomib) alone or in combination with another chemotherapeutic agent) to increase the likelihood that a patient will have a slower time to death comprising determining the expression profile of a predictive marker or predictive marker set; and identifying and/or advising that a proteasome inhibition therapeutic agent
  • determining whether a proteasome inhibitor therapy can be used to increase the likelihood that a patient will have a slower time to death comprising obtaining a sample of tumor cells, evaluating the expression of one or more individual markers or a marker set, both in tumor cells exposed to the agent and in tumor cells that have not been exposed to the proteasome inhibition therapy; and identifying and/or advising that an agent is or is not appropriate or that a dosing or administration schedule is appropriate or is not appropriate to treat the tumor based on the evaluation.
  • the invention provides a method for determining whether a glucocorticoid regimen (e.g., glucocorticoidal steroid agent (e.g., dexamethasone) alone or in combination with another chemotherapeutic agent) can be used to increase the likelihood that a patient will have a slower time to death comprising determining the expression profile of a predictive marker or predictive marker set; and identifying and/or advising that a glucocorticoid therapeutic agent is or is not appropriate or that a dosing or administration schedule is appropriate or is not appropriate to increase the likelihood that a patient will have a slower time to death based on the expression profile.
  • a glucocorticoid regimen e.g., glucocorticoidal steroid agent (e.g., dexamethasone) alone or in combination with another chemotherapeutic agent
  • a glucocorticoid regimen e.g., glucocorticoidal steroid agent (e.g., de
  • a glucocorticoid therapy can be used to increase the likelihood a patient will have a slower time to death, comprising obtaining a sample of tumor cells, evaluating the expression of one or more individual markers or a marker set, both in tumor cells exposed to the agent and in tumor cells that have not been exposed to the glucocorticoid therapy; and identifying and/or advising that an agent is or is not appropriate or that a dosing or administration schedule is appropriate or is not appropriate to treat the tumor based on the evaluation.
  • a proteasome inhibition therapy and/or glucocorticoid therapy is determined appropriate to treat the tumor when the expression profile of the predictive marker or predictive marker set demonstrates a long term survivor according to the expression profile of the predictive markers in the presence of the agent.
  • a proteosome inhibition therapy and/or glucocorticoid therapy is determined to be appropriate to treat the tumor but a more aggressive dose and/or administration schedule when the expression profile of the predicted marker or predictive marker set demonstrates a short term survivor.
  • the invention also provides a method for determining whether treatment with an proteasome inhibitor therapy and/or glucocorticoid therapy should be initiated in a patient selected from a multiple myeloma patient, a lymphoma patient, a leukemia patient, a lung cancer patient, a breast cancer patient, and an ovarian cancer patient , a prostate cancer patient, a colon cancer patient, a kidney cancer patient, and a liver cancer patient; comprising obtaining one or more samples, followed by determining the level of expression of one or more markers which correspond to markers identified in any of Table 1 and Table 2 in the sample; and initiating proteasome inhibitor therapy when the expression profile of the predictive markers identified in any one of Table 1 and Table 2 is indicative of enhanced survival time with such treatment.
  • the treatment is not initiated, or is initiated at a more aggressive dose and/or administration schedule when the expression profile of the predictive markers identified in any one of Table 1 and Table 2 is indicative of a predicted shorter survival time with the treatment.
  • the identified short term and long term survival markers can be used as pharmacodynamic markers to assess whether the tumor has changed in a way to affect predicted survival time.
  • the markers can assess whether the tumor has become refractory to an ongoing treatment (e.g., a proteasome inhibition therapy and/or glucocorticoid therapy).
  • an ongoing treatment e.g., a proteasome inhibition therapy and/or glucocorticoid therapy.
  • the expression profile of the tumor cells will change: the level or relative expression of one or more of the predictive markers (e.g., those predictive markers identified in Table 1 and Table 2) such that the expression profile represents a short term survivor patient.
  • the invention provides methods for determining or advising whether a cancer therapy comprising proteasome inhibition therapy regimen and/or glucocorticoid therapy regimen should be continued in a cancer patient, comprising determining the expression of at least one predictive marker or a marker set, wherein the markers are selected from those set forth in any of Table 1 and Table 2, in a tumor sample of a patient exposed to a proteasome inhibition therapy and/or glucocorticoid therapy; and continuing treatment when the expression profile of the marker or marker set demonstrates that the patient is a long term survivor.
  • the invention provides methods for determining or advising whether a cancer therapy comprising proteasome inhibition therapy regimen and/or glucocorticoid therapy regimen should be continued in a cancer patient, comprising determining the expression of at least one predictive marker or a marker set, wherein the markers are selected from those set forth in any of Table 1 and Table 2, in a tumor sample of a patient exposed to a proteasome inhibition therapy and/or glucocorticoid therapy; and altering the therapy to an alternative agent or agents other than proteosome inhibitors and/or glucocorticoids that is expected to have a similar effect on survival when the expression profile of the marker or marker set demonstrates that the patient is a long term survivor.
  • the invention provides methods for determining or advising whether a cancer therapy comprising proteasome inhibition therapy regimen and/or glucocorticoid therapy regimen should be continued in a cancer patient, comprising determining the expression of at least one predictive marker or a marker set, wherein the markers are selected from those set forth in any of Table 1 and Table 2, in a tumor sample of a patient exposed to a proteasome inhibition therapy and/or glucocorticoid therapy; and altering the treatment, e.g., administer in conjunction with other chemotherapeutic agents and/or apply a more aggressive dose and/or administration schedule when the expression profile of the marker or marker set demonstrates that the patient is a short term survivor.
  • the invention provides methods for determining whether a proteasome inhibition therapy and/or glucocorticoid therapy should be discontinued in a cancer patient, comprising determining the expression of at least one predictive marker or a predictive marker set, wherein the markers are selected from those set forth in any of Table 1 and Table 2 in a tumor sample of a patient exposed to a proteasome inhibition therapy and/or glucocorticoid therapy; and discontinuing or altering treatment when the expression profile of the markers identified in any one of Table 1 and Table 2 demonstrates that the patient is a short term survivor.
  • a patient refers to any subject having cancer.
  • the subject may be a human patient undergoing proteasome inhibition (e.g., bortezomib or other related agent) and/or glucocorticoid (e.g., dexamethasone) therapy using a sole therapeutic agent.
  • the subject may be a human patient undergoing proteasome inhibition (e.g., bortezomib or other related agent) and/or glucocorticoid (e.g., dexamethasone) therapy using a therapeutic agent in conjunction with another agent (e.g., a chemotherapy treatment).
  • the present invention also includes comparing two or more samples obtained from a patient undergoing anti-cancer treatment including proteasome inhibition therapy and/or glucocorticoid therapy.
  • a first sample from the patient prior to beginning therapy and one or more samples during . treatment In such a use, a baseline of expression prior to therapy is determined, then changes in the baseline state of expression is monitored during the course of therapy. Alternatively, two or more successive samples obtained during treatment can be used without the need of a pre- treatment baseline sample. In such a use, the first sample obtained from the subject is used as a baseline for determining whether the expression of a particular marker or marker set is increasing or decreasing].
  • two or more samples from a patient are examined.
  • three or more successively obtained samples are used, including at least one pretreatment sample.
  • the invention provides methods for determining whether treatment with a proteasome inhibitor therapy regimen should be continued in a patient selected from a multiple myeloma patient, a lymphoma patient, a leukemia patient, a lung cancer patient, a breast cancer patient, and an ovarian cancer patient , a prostate cancer patient, a colon cancer patient, a kidney cancer patient, and a liver cancer patient; comprising obtaining two or more samples of tumor cells from a patient at different times during the course of a proteasome inhibition therapy regimen, followed by evaluating the expression of one or more markers which correspond to markers identified in any of Table 1 and Table 2 in the two or more samples; and continuing the treatment when the expression profile of the predictive markers identified in any one of Table 1, and Table 2 is indicative of a long term or short term survivor during the course of the treatment.
  • a proteasome inhibition therapy and regimen is determined appropriate to treat the patient when the expression profile of the predictive marker or predictive marker set more typifies long term survival or less typifies short term survival according
  • the treatment is discontinued when the expression profile of the marker set more typifies short term survival and/or less typtif ⁇ es long term survival during the course of treatment.
  • Certain aspects of the invention relate to methods of treatment and/or diagnosis of a patient with cancer utilizing samples.
  • the source of the cancer cells used in the present methods will be based on how the method of the present invention is being used. For example, if the method is being used to determine whether a patient's cancer can be treated with an agent, or a combination of agents, or a particular dosage and/or administration therapy regimen then the preferred source of sample will be cancer cells obtained from a tumor from the patient, e.g., a tumor biopsy (including a solid or a liquid tumor), a blood sample, a plasma sample, a urine sample, a saliva sample, a lymph sample or other sample can be used.
  • a tumor biopsy including a solid or a liquid tumor
  • a sample obtained from a tumor can be enriched for tumor cells to increase the specificity of the analysis.
  • a variety of methods known in the art can be used to enrich for tumor cells, including differential centrifugation,, fluorescence cell sorting analysis (FACS), isolating cells based on growth independent of substrate attachment, binding to a selection agent, e.g. to an antibody to a tumor marker and furthermore attaching the antibody and thus the bound tumor cell to a solid support, etc, or conversely, an antibody to a marker on a non-tumor cell, e.g.
  • CD14 monoocytes
  • CD2 T and NK cells
  • CD33 myeloid progenitors and monocytes
  • CD41 platelets and megakaryocytes
  • CD45RA na ⁇ ve B and T cells
  • CD66b granulocytes
  • a cancer cell line similar to the type of cancer being treated can be assayed. For example, if multiple myeloma is being treated, then a myeloma cell line can be used. If the method is being used to predict or monitor the effectiveness of a therapeutic protocol, then a tissue or blood sample from a patient being treated is a preferred source.
  • a skilled artisan can readily select and obtain the appropriate cancer cells that are used in the present method.
  • sources such as The National Cancer Institute, Bethesda, MD, for the NCI-60 cancer cell panel, are preferred.
  • Other cell lines e.g. from American Type Culture Collection (ATCC®), Manassas, VA), e.g. myeloma cell lines (e.g., RPMI-8226 or U266) or cell lines of other tumors, e.g.
  • B -cell lymphoma BC-3
  • colon tumor HCT 116
  • breast tumor MDA-MB-231
  • cervical tumor HeLa
  • lung tumor A549)
  • melanoma A375
  • prostate tumor 22RvI
  • normal cells e.g. from kidney (HEK293)
  • standard biopsy methods such as a needle biopsy, can be employed.
  • the expression level of markers can be evaluated in other tissue types including disorders of related hematological cell types, including, e.g., Waldenstroms macrogobulinemia, Myelodysplastic syndrome and other hematological cancers including lymphomas, leukemias, as well as tumors of various solid tissues. It will thus be appreciated that cells from other hematologic malignancies including, e.g., B-cell Lymphomas, Non-Hodgkins Lymphoma, Waldenstrom's syndrome, or other leukemias will be useful in the methods of the present invention.
  • disorders of related hematological cell types including, e.g., Waldenstroms macrogobulinemia, Myelodysplastic syndrome and other hematological cancers including lymphomas, leukemias, as well as tumors of various solid tissues.
  • cells from other hematologic malignancies including, e.g., B-cell Lymphomas, Non-Hodgkins Lymphoma, Waldenstrom's syndrome, or
  • the predictive markers predicting disease aggressiveness as well as short term and long term survival to agents such as proteasome inhibition therapeutic agents in solid tumors can also be useful in the methods of the present invention.
  • the samples used will be from similar tumors or from noncancerous cells of the same tissue origin as the tumor in question.
  • the choice of the cell source is dependent on the use of the relative expression level data. For example, using tumors of similar types for obtaining a mean expression score allows for the identification of extreme cases of short term or long term survival. Using expression found in normal tissues as a mean expression score aids in validating whether the short term/long term survival marker or marker set assayed is tumor specific (versus normal cells). Such a later use is particularly important in identifying whether a short term or long term survivor marker or marker set can serve as a target marker or marker set.
  • the mean expression value can be revised, providing improved relative expression values based on accumulated data.
  • markers include gene array/chip technology, RT-PCR, in-situ hybridization, immunohistochemistry, immunoblotting, FISH (fluoresence in-situ hybridization), FACS analyses, northern blot, southern blot or cytogenetic analyses.
  • FISH fluoresence in-situ hybridization
  • FACS analyses northern blot, southern blot or cytogenetic analyses.
  • a skilled artisan can select from these or other appropriate and available methods based on the nature of the marker(s), tissue sample and disease in question. Different methods or combinations of methods could be appropriate in different cases or, for instance in different solid or hematological tumor types.
  • the expression of predictive marker or markers identified in Table 1 and Table 2 is detected by measuring mRNA which corresponds to the predictive marker or marker set.
  • the expression of markers which correspond to markers or marker sets identified in Table 1, and Table 2 is detected by measuring protein which corresponds to the marker or marker set.
  • An exemplary method for detecting the presence or absence of a nucleic acid or polypeptide corresponding to a marker of the invention in a biological sample involves obtaining a biological sample (e.g. a tumor sample) from a test subject and contacting the biological sample with a compound or an agent capable of detecting the polypeptide or nucleic acid (e.g., mRNA, genomic DNA, or cDNA).
  • a biological sample e.g. a tumor sample
  • a compound or an agent capable of detecting the polypeptide or nucleic acid e.g., mRNA, genomic DNA, or cDNA.
  • the detection methods of the invention can thus be used to detect mRNA, protein, cDNA, or genomic DNA, for example, in a biological sample in vitro as well as in vivo.
  • in vitro techniques for detection of mRNA include Northern hybridizations, in situ hybridizations, and TAQMAN® gene expression assays (Applied Biosystems, Foster City, CA) under GLP approved laboratory conditions.
  • in vitro techniques for detection of a polypeptide corresponding to a marker of the invention include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence.
  • In vitro techniques for detection of genomic DNA include Southern hybridizations.
  • in vivo techniques for detection of a polypeptide corresponding to a marker of the invention include introducing into a subject a labeled antibody directed against the polypeptide.
  • the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
  • a general principle of such diagnostic and prognostic assays involves preparing a sample or reaction mixture that may contain a marker, and a probe, under appropriate conditions and for a time sufficient to allow the marker and probe to interact and bind, thus forming a complex that can be removed and/or detected in the reaction mixture.
  • These assays can be conducted in a variety of ways.
  • one method to conduct such an assay would involve anchoring the marker or probe onto a solid phase support, also referred to as a substrate, and detecting target marker/probe complexes anchored on the solid phase at the end of the reaction.
  • a sample from a subject which is to be assayed for presence and/or concentration of marker, can be anchored onto a carrier or solid phase support.
  • the reverse situation is possible, in which the probe can be anchored to a solid phase and a sample from a subject can be allowed to react as an unanchored component of the assay.
  • One example of such an example includes use of an array or chip which contains a predictive marker or marker set anchored for expression analysis of the sample.
  • biotinylated assay components can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilized in the wells of streptavidin- coated 96 well plates (Pierce Chemical).
  • biotinylation kit N-hydroxy-succinimide
  • Pierce Chemicals Pierce Chemicals, Rockford, IL
  • streptavidin- coated 96 well plates Piereptavidin- coated 96 well plates
  • suitable carriers or solid phase supports for such assays include any material capable of binding the class of molecule to which the marker or probe belongs.
  • Well-known supports or carriers include, but are not limited to, glass, polystyrene, nylon, polypropylene, nylon, polyethylene, dextran, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite.
  • the non- immobilized component is added to the solid phase upon which the second component is anchored.
  • uncomplexed components may be removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized upon the solid phase.
  • the detection of marker/probe complexes anchored to the solid phase can be accomplished in a number of methods outlined herein.
  • the probe when the probe is the unanchored assay component, can be labeled for the purpose of detection and readout of the assay, either directly or indirectly, with detectable labels discussed herein and which are well-known to one skilled in the art.
  • marker/probe complex formation without further manipulation or labeling of either component (marker or probe), for example by utilizing the technique of fluorescence energy transfer (see, for example, Lakowicz et al, U.S. Patent No. 5,631,169; Stavrianopoulos, et al., U.S. Patent No. 4,868,103).
  • a fluorophore label on the first, 'donor' molecule is selected such that, upon excitation with incident light of appropriate wavelength, its emitted fluorescent energy will be absorbed by a fluorescent label on a second 'acceptor' molecule, which in tum is able to fluoresce due to the absorbed energy.
  • the 'donor' protein molecule may simply utilize the natural fluorescent energy of tryptophan residues. Labels are chosen that emit different wavelengths of light, such that the 'acceptor' molecule label may be differentiated from that of the 'donor'. Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, spatial relationships between the molecules can be assessed. Li a situation in which binding occurs between the molecules, the fluorescent emission of the 'acceptor' molecule label in the assay should be maximal. An FET binding event can be conveniently measured through standard fluorometric detection means well known in the art ( e.g., using a fluorimeter).
  • determination of the ability of a probe to recognize a marker can be accomplished without labeling either assay component (probe or marker) by utilizing a technology such as real-time Biomolecular Interaction Analysis (BIA) (see, e.g., Sjolander, S. and Urbaniczky, C, 1991, Anal Chem. 63:2338-2345 and Szabo et al, 1995, Curr. Opin. Struct. Biol. 5:699-705).
  • BIOA Biomolecular Interaction Analysis
  • surface plasmon resonance is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIACORETM).
  • analogous diagnostic and prognostic assays can be conducted with marker and probe as solutes in a liquid phase.
  • the complexed marker and probe are separated from uncomplexed components by any of a number of standard techniques, including but not limited to: differential centrifugation, chromatography, electrophoresis and immunoprecipitation.
  • marker/probe complexes may be separated from uncomplexed assay components through a series of centrifugal steps, due to the different sedimentation equilibria of complexes based on their different sizes and densities (see, for example, Rivas, G., and Minton, A.P., 1993, Trends Biochem ScL 18(8):284-7).
  • Standard chromatographic techniques may also be utilized to separate complexed molecules from uncomplexed ones. For example, gel filtration chromatography separates molecules based on size, and through the utilization of an appropriate gel filtration resin in a column format, for example, the relatively larger complex may be separated from the relatively smaller uncomplexed components.
  • the relatively different charge properties of the marker/probe complex as compared to the uncomplexed components may be exploited to differentiate the complex from uncomplexed components, for example through the utilization of ion-exchange chromatography resins.
  • ion-exchange chromatography resins Such resins and chromatographic techniques are well known to one skilled in the art (see, e.g., Heegaard, N.H., 1998, J. MoI. Recognit. Winter ll(l-6):141-8; Hage, D.S., and Tweed, S.A. J Chromatogr B Biomed Sci Appl 1997 Oct 10;699(l-2):499-525).
  • Gel electrophoresis may also be employed to separate complexed assay components from unbound components (see, e.g., Ausubel et al, ed., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1987- 1999). Ih this technique, protein or nucleic acid complexes are separated based on size or charge, for example. In order to maintain the binding interaction during the electrophoretic process, non- denaturing gel matrix materials and conditions in the absence of reducing agent are typically preferred. Appropriate conditions to the particular assay and components thereof will be well known to one skilled in the art.
  • the level of mRNA corresponding to the marker can be determined both by in situ and by in vitro formats in a biological sample using methods known in the art.
  • biological sample is intended to include tissues, cells, biological fluids and isolates thereof, isolated from a subject, as well as tissues, cells and fluids present within a subject.
  • Many expression detection methods use isolated RNA.
  • any RNA isolation technique that does not select against the isolation of mRNA can be utilized for the purification of RNA from tumor cells (see, e.g., Ausubel et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, New York 1987-1999).
  • RNA isolation process of Chomczynski (1989, U.S. Patent No. 4,843,155).
  • the nucleic acid probe can be, for example, a full-length cDNA, or a portion thereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions, e.g., hybridize under conditions of hybridization of 6X SSC at 45°C and washing in 0.2 X SSC, 0.1% SDS at 65°C, to a mRNA or genomic DNA encoding a marker of the present invention.
  • Other suitable probes for use in the diagnostic assays of the invention are described herein. Hybridization of an mRNA with the probe indicates that the marker in question is being expressed.
  • the mRNA is immobilized on a solid surface and contacted with a probe, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose.
  • the probe(s) are immobilized on a solid surface and the mRNA is contacted with the probe(s), for example, in an AFFYMETRIX® gene chip array.
  • a skilled artisan can readily adapt known mRNA detection methods for use in detecting the level of mRNA encoded by the markers of the present invention.
  • An alternative method for determining the level of mRNA corresponding to a marker of the present invention in a sample involves the process of nucleic acid amplification, e.g., by rtPCR (the experimental description set forth in Mullis, 1987, U.S. Patent No. 4,683,202), ligase chain reaction (Barany, 1991, Proc. Natl. Acad. ScL USA, 88:189-193), self sustained sequence replication (Guatelli et al, 1990, Proc. Natl. Acad. ScL USA 87:1874-1878), transcriptional amplification system (Kwoh et al., 1989, Proc. Natl. Acad.
  • amplification primers are defined as being a pair of nucleic acid molecules that can anneal to 5' or 3' regions of a gene (plus and minus strands, respectively, or vice-versa) and contain a short region in between.
  • amplification primers are from about 10 to 30 nucleotides in length and flank a region from about 50 to 200 nucleotides in length. Under appropriate conditions and with appropriate reagents, such primers permit the amplification of a nucleic acid molecule comprising the nucleotide sequence flanked by the primers.
  • mRNA does not need to be isolated from the cancer cells prior to detection.
  • a cell or tissue sample is prepared/processed using known histological methods. The sample is then immobilized on a support, typically a glass slide, and then contacted with a probe that can hybridize to mRNA that encodes the marker.
  • determinations may be based on the normalized expression level of the marker. Expression levels are normalized by correcting the absolute expression level of a marker by comparing its expression to the expression of a reference gene that is not a marker, e.g., a housekeeping gene that is constitutively expressed.
  • Suitable genes for normalization include housekeeping genes such as the actin gene, or epithelial cell-specific genes. This normalization allows the comparison of the expression level in one sample, e.g., a patient sample, to another sample, e.g., a non- cancer sample, or between samples from different sources.
  • the expression level can be provided as a relative expression level. To determine a relative expression level of a marker, the level of expression of the marker is determined for 10 or more samples of normal versus cancer cell isolates, preferably 50 or more samples, prior to the determination of the expression level for the sample in question. The mean expression level of each of the markers and marker sets assayed in the larger number of samples is determined and this is used as a baseline expression level for the marker.
  • a polypeptide corresponding to a marker is detected.
  • a preferred agent for detecting a polypeptide of the invention is an antibody capable of binding to a polypeptide corresponding to a marker of the invention, preferably an antibody with a detectable label.
  • Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab') 2 ) can be used.
  • labeled with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (Le., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled.
  • indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin. Additional examples of detectable substances are detailed in a later section.
  • a variety of formats can be employed to determine whether a sample contains a protein that binds to a given antibody.
  • formats include, but are not limited to, enzyme immunoassay (EIA), radioimmunoassay (RIA), Western blot analysis and enzyme linked immunoabsorbant assay (ELISA).
  • EIA enzyme immunoassay
  • RIA radioimmunoassay
  • ELISA enzyme linked immunoabsorbant assay
  • antibodies, or antibody fragments can be used in methods such as Western blots or immunofluorescence techniques to detect the expressed proteins.
  • Suitable solid phase supports or carriers include any support capable of binding an antigen or an antibody.
  • Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite.
  • One skilled in the art will know many other suitable carriers for binding antibody or antigen, and will be able to adapt such support for use with the present invention.
  • protein isolated from tumor cells can be run on a polyacrylamide gel electrophoresis and immobilized onto a solid phase support such as nitrocellulose.
  • the support can then be washed with suitable buffers followed by treatment with the detectably labeled antibody.
  • the solid phase support can then be washed with the buffer a second time to remove unbound antibody.
  • the amount of bound label on the solid support can then be detected by conventional means.
  • Another method for determining the level of a polypeptide corresponding to a marker is mass spectrometry.
  • intact proteins or peptides can be analyzed from a sample, e.g., a tumor biopsy (including a solid or a liquid tumor), a blood sample, a plasma sample, a urine sample, a saliva sample, a lymph sample or other sample, containing one or more polypeptide markers.
  • the method can further include treating the sample to lower the amounts of abundant proteins, e.g. serum albumin, to increase the sensitivity of the method.
  • liquid chromatography can be used to fractionate the sample so portions of the sample can be analyzed separately by mass spectrometry.
  • the steps can be performed in separate systems or in a combined liquid chromatography/mass spectrometry system (LC/MS, see for example, Liao, et al. Arthritis Rheum. 50:3792-3803 (2004)).
  • the mass spectrometry system also can be in tandem (MS/MS) mode.
  • the charge state distribution of the protein or peptide mixture can be acquired over one or multiple scans and analyzed by statistical methods, e.g. using the retention time and mass-to-charge ratio (m/z) in the LC/MS system, to identify proteins expressed at statistically significant levels differentially in samples from patients responsive or non-responsive to proteasome inhibition and/or glucocorticoid therapy.
  • mass spectrometers which can be used are an ion trap system (ThermoFinnigan, San Jose, CA) or a quadrupole time-of-flight mass spectrometer (Applied Biosystems, Foster City, CA).
  • the method can further include the step of peptide mass fingerprinting, e.g. in a matrix-assisted laser desorption ionization with time- of-flight (MALDI-TOF) mass spectrometry method.
  • the method can further include the step of sequencing one or.more of the tryptic peptides. Results of this method can be used to identify proteins from primary sequence databases, e.g. maintained by the National Center for Biotechnology Information, Bethesda, MD, or the Swiss Institute for Bioinformatics, Geneva, Switzerland, and based on mass spectrometry tryptic peptide m/z base peaks.
  • Electronic apparatus including readable arrays comprising at least one predictive marker of the present invention is also contemplated for use in conjunction with the methods of the invention.
  • electronic apparatus readable media refers to any suitable medium for storing, holding or containing data or information that can be read and accessed directly by an electronic apparatus.
  • electronic apparatus is intended to include any suitable computing or processing apparatus or other device configured or adapted for storing data or information.
  • Examples of electronic apparatus suitable for use with the present invention and monitoring of the recorded information include stand-alone computing apparatus; networks, including a local area network (LAN), a wide area network (WAN) Internet, Intranet, and Extranet; electronic appliances such as personal digital assistants (PDAs), cellular phone, pager and the like; and local and distributed processing systems.
  • LAN local area network
  • WAN wide area network
  • Extranet Internet
  • PDAs personal digital assistants
  • recording information on the electronic apparatus readable medium Those skilled in the art can readily adopt any of the presently known methods for recording information on known media to generate manufactures comprising the markers of the present invention.
  • microarray systems are well known and used in the art for assessment of samples, whether by assessment gene expression (e.g., RNA detection, protein detection), or metabolite production, for example.
  • Microarrays for use according to the invention include one or more probes of predictive marker(s) of the invention characteristic of response and/or non-response to a therapeutic regimen as described herein.
  • the microarray comprises one or more probes corresponding to one or more of markers selected from the group consisting of markers which demonstrate increased expression in short term survivors, and genes which demonstrate increased expression in long term survivors in patients.
  • a number of different microarray configurations and methods for their production are known to those of skill in the art and are disclosed, for example, in U.S. Pat.
  • a phage-epitope microarray can be used to identify one or more proteins in a sample based on whether the protein or proteins induce autoantibodies in the patient (Bradford et al. Urol. Oncol. 24:237-242 (2006)).
  • a microarray thus comprises one or more probes corresponding to one or more predictive markers identified in Table 1 and Table 2.
  • the microarray may comprise probes corresponding to, for example, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 75, at least 100, at least 200, at least 300, or at least 400 predictive markers of the invention predictive of short term or long term survival of a cancer patient.
  • the microarray may comprise probes corresponding to one or more predictive markers as set forth herein. Still further, the microarray may comprise complete marker sets as set forth herein and which may be selected and compiled according to the methods set forth herein.
  • the microarray can be used to assay expression of one or more predictive markers or predictive marker sets in the array. In one example, the array can be used to assay more than one predictive marker or marker set expression in a sample to ascertain an expression profile of markers in the array. In this manner, up to about 44,000 markers can be simultaneously assayed for expression. This allows a profile to be developed showing a battery of markers specifically expressed in one or more samples. Still further, this allows a profile to be developed to assess overall survival.
  • the array is also useful for ascertaining differential expression patterns of one or more markers in normal and abnormal (e.g., sample, e.g., tumor) cells. This provides a battery of predictive markers that could serve as a tool for ease of identification of short term or long term survival patients. Further, the array is useful for ascertaining expression of reference markers for reference expression levels. In another example, the array can be used to monitor the time course of expression of one or more predictive markers in the array.
  • the invention allows the quantitation of marker expression.
  • predictive markers can be grouped on the basis of marker sets or short term or long term survival indications by the level of expression in the sample. This is useful, for example, in ascertaining the short term or long term survival indication of the sample by virtue of scoring the expression levels according to the methods provided herein.
  • the array is also useful for ascertaining the effect of the expression of a marker on the expression of other predictive markers in the same cell or in different cells. This provides, for example, a selection of alternate molecular targets for therapeutic intervention if patient is predicted to be a short term survivor.
  • the invention provides a test kit for monitoring the efficacy of a compound or therapeutic in a sample.
  • the kit may comprise a labeled probe capable of detecting a polypeptide or an mRNA encoding a polypeptide corresponding to a marker of the invention in a biological sample and means for determining the amount of the polypeptide or mRNA in the sample ⁇ e.g., an antibody which binds the polypeptide or an oligonucleotide probe which binds to DNA or mRNA encoding the polypeptide).
  • Kits may further include instructions for use of the provided kits and interpreting the results obtained using the kit; additional reagents for preparation of probes for use in the methods provided; and detectable label, alone or conjugated to the provided probe(s).
  • the kit can comprise, for example: (1) a first antibody
  • a polypeptide corresponding to a marker of the invention
  • a second, different antibody which binds to either the polypeptide or the first antibody and is conjugated to a detectable label
  • the kit can comprise, for example: (1) an oligonucleotide, e.g., a detectably labeled oligonucleotide, which hybridizes to a nucleic acid sequence encoding a polypeptide corresponding to a marker of the invention; (2) a pair of primers useful for amplifying a nucleic acid molecule corresponding to a marker of the invention; or (3) a marker set comprising oligonucleotides which hybridize to at least two nucleic acid sequences encoding polypeptide predictive markers of the invention.
  • the kit can also comprise, e.g., a buffering agent, a preservative, or a protein stabilizing agent.
  • the kit can further comprise components necessary for detecting the detectable label (e.g., an enzyme or a substrate).
  • the kit can comprise a marker set array or chip for use in detecting the predictive markers.
  • the kit can also contain a reference sample or a series of reference samples which can be assayed and compared to the test sample. Each component of the kit can be enclosed within an individual container and all of the various containers can be within a single package, along with instructions for interpreting the results of the assays performed using the kit.
  • the markers and marker sets of the present invention assess the likelihood of short or long term survival in cancer patients, e.g., patients having multiple myeloma. Using this prediction, cancer therapies can be evaluated to design a therapy regimen best suitable for patients in either category.
  • Therapeutic agents for use in the methods of the invention include a class of therapeutic agents known as proteosome inhibitors
  • proteasome inhibitor refers to any substance which directly inhibits enzymatic activity of the 2OS or 26S proteasome in vitro or in vivo.
  • the proteasome inhibitor is a peptidyl boronic acid. Examples of peptidyl boronic acid proteasome inhibitors suitable for use in the methods of the invention are disclosed in Adams et al., U.S. Patent Nos.
  • the peptidyl boronic acid proteasome inhibitor is selected from the group consisting of: N (4 morpholine)carbonyl-.beta.-(l-naphthyl)-L-alanine-L-leucine boronic acid; N (8 quinoline)sulfonyl-.beta.-(l-naphthyl)-L-alanine-L-aIanine-L-leucine boronic acid; N (pyrazine)carbonyl-L-phenylalanine-L-leucine boronic acid, and N (4 morpholine)-carbonyl-[O- (2-pyridylmethyl)]-L-tyrosine-L-leucine boronic acid.
  • the proteasome inhibitor is N (pyrazine)carbonyl-L-phenylalanine-L-leucine boronic acid (bortezomib; VELCADE®; formerly known as MLN341 or PS-341).
  • Additional peptidyl boronic acid proteasome inhibitors are disclosed in Si man et al., international patent publication WO 99/30707; Bernareggi et al., international patent publication WO 05/021558; Chatterjee et al., international patent publication WO 05/016859; Furet et al., U.S. patent publication 2004/0167337; Furet et al., international patent publication 02/096933; Attwood et al., U.S. Patent No. 6,018,020 (2000); Magde et al., international patent publication WO 04/022070; and Purandare and Laing, international patent publication WO 04/064755.
  • proteasome inhibitors include peptide aldehyde proteasome inhibitors, such as those disclosed in Stein et al., U.S. Patent No. 5,693,617 (1997); Siman et al., international patent publication WO 91/13904; Iqbal et al., J. Med. Chem. 38:2276-2277 (1995); and Iinuma et al., international patent publication WO 05/105826, each of which is hereby incorporated by reference in its entirety.
  • proteasome inhibitors include peptidyl epoxy ketone proteasome inhibitors, examples of which are disclosed in Crews et al., U.S. Patent No.
  • proteasome inhibitors include alpha-ketoamide proteasome inhibitors, examples of which are disclosed in Chatterjee and Mallamo, U.S. Patent Nos. 6,310,057 (2001) and 6,096,778 (2000); and Wang et al., U.S. Patent Nos. 6,075,150 (2000) and 6,781,000 (2004), each of which is hereby incorporated by reference in its entirety.
  • Additional proteasome inhibitors include peptidyl vinyl ester proteasome inhibitors, such as those disclosed in Marastoni et al., J. Med. Chem.
  • proteasome inhibitors include azapeptoids and hydrazinopeptoids, such as those disclosed in Bouget et al., Bioorg. Med. Chem. 11:4881 (2003); Baudy-Floc'h et al., international patent publication WO 05/030707; and Bonnemains et al., international patent publication WO 03/018557, each of which is hereby incorporated by reference in its entirety.
  • proteasome inhibitors include peptide derivatives, such as those disclosed in Furet et al., U.S. patent publication 2003/0166572, and efrapeptin oligopeptides, such as those disclosed in Papathanassiu, international patent publication WO 05/115431, each of which is hereby incorporated by reference in its entirety.
  • proteasome inhibitors include lactacystin and salinosporamide and analogs thereof, which have been disclosed in Fenteany et al., U.S. Patent Nos. 5,756,764 (1998), 6,147,223 (2000), 6,335,358 (2002), and 6,645,999 (2003); Fenteany et al., Proc. Natl. Acad. Sci. USA (1994) 91:3358; Fenical et al., international patent publication WO 05/003137; Palladino et al., international patent publication WO 05/002572; Stadler et al., international patent publication WO 04/071382; Xiao and Patel, U.S. patent publication 2005/023162; and Corey, international patent publication WO 05/099687, each of which is hereby incorporated by reference in its entirety.
  • proteasome inhibitors include polyphenol proteasome inhibitors, such as those disclosed in Nam et al., J. Biol. Chem.
  • Additional therapeutic agents for use in the methods of the invention comprise a known class of therapeutic agents comprising glucocorticoid steroids.
  • Glucocorticoid therapy generally comprises at least one glucocorticoid agent (e.g., dexamethasone).
  • the agent used in methods of the invention is a glucocorticoid agent.
  • a glucocorticoid utilized in the treatment of multiple myeloma patients as well as other cancer therapies is dexamethasone.
  • Additional glucocorticoids utilized in treatment of hematological and combination therapy in solid tumors include hydrocortisone, predisolone, prednisone, and triamcinolone.
  • Glucocorticoid therapy regimens can be used alone, or can be used in conjunction with additional chemotherapeutic agents.
  • Chemotherapeutic agents are known in the art and described in further detail herein. Examples of chemotherapeutic agents are set forth in Table A.
  • proteasome inhibition therapy new classes of cancer therapies may be combined with glucocorticoid therapy regimens as they are developed.
  • the methods of the invention include combination of proteasome inhibition therapy with glucocorticoid therapy, either alone, or in conjunction with further agents.
  • proteasome inhibition therapy regimen and/or glucocorticoid therapy regimen can include additional agents in addition to proteasome inhibition agents, including chemotherapeutic agents.
  • a "chemotherapeutic agent” is intended to include chemical reagents which inhibit the growth of proliferating cells or tissues wherein the growth of such cells or tissues is undesirable.
  • Chemotherapeutic agents such as anti-metabolic agents, e.g., Ara AC, 5-FU and methotrexate, antimitotic agents, e.g., taxane, vinblastine and vincristine, alkylating agents, e.g., melphanlan, Carmustine (BCNU) and nitrogen mustard, Topoisomerase II inhibitors, e.g., VW-26, topotecan and Bleomycin, strand-breaking agents, e.g., doxorubicin and Mitoxantrone (DHAD), cross-linking agents, e.g., cisplatin and carboplatin (CBDCA), radiation and ultraviolet light.
  • anti-metabolic agents e.g., Ara AC, 5-FU and methotrexate
  • antimitotic agents e.g., taxane, vinblastine and vincristine
  • alkylating agents e.g., melphanlan, Carmustine (BCNU) and nitrogen mustard
  • the agent is a proteasome inhibitor (e.g., bortezomib or other related compounds).are well known in the art (see e.g., Gilman A. G., et aL, The Pharmacological Basis of Therapeutics, 8th Ed., Sec 12: 1202-1263 (1990)), and are typically used to treat neoplastic diseases.
  • the chemotherapeutic agents generally employed in chemotherapy treatments are listed below in Table A. [00137]
  • the agents tested in the present methods can be a single agent or a combination of agents.
  • the present methods can be used to determine whether a single chemotherapeutic agent, such as methotrexate, can be used to treat a cancer or whether a combination of two or more agents can be used in combination with a proteasome inhibitor(e.g., bortezomib) and/or a glucocorticoid agent (e.g., dexamethasone).
  • a proteasome inhibitor e.g., bortezomib
  • a glucocorticoid agent e.g., dexamethasone
  • Preferred combinations will include agents that have different mechanisms of action, e.g., the use of an anti-mitotic agent in combination with an alkylating agent and a proteasome inhibitor.
  • the agents disclosed herein may be administered by any route, including intradermally, subcutaneously, orally, intraarterially or intravenously. Preferably, administration will be by the intravenous route. Preferably parenteral administration may be provided in a bolus or by infusion.
  • the concentration of a disclosed compound in a pharmaceutically acceptable mixture will vary depending on several factors, including the dosage of the compound to be administered, the pharmacokinetic characteristics of the compound(s) employed, and the route of administration.
  • the agent may be administered in a single dose or in repeat doses. Treatments may be administered daily or more frequently depending upon a number of factors, including the overall health of a patient, and the formulation and route of administration of the selected compound(s).
  • One aspect of the invention pertains to isolated nucleic acid molecules that correspond to a predictive marker of the invention, including nucleic acids which encode a polypeptide corresponding to a predictive marker of the invention or a portion of such a polypeptide.
  • isolated nucleic acids of the invention also include nucleic acid molecules sufficient for use as hybridization probes to identify nucleic acid molecules that correspond to a predictive marker of the invention, including nucleic acids which encode a polypeptide corresponding to a predictive marker of the invention, and fragments of such nucleic acid molecules, e.g., those suitable for use as PCR primers for the amplification or mutation of nucleic acid molecules.
  • nucleic acid molecule is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs.
  • the nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.
  • a nucleic acid molecule of the present invention e.g., a nucleic acid encoding a protein corresponding to a marker listed in any one of Table 1 and Table 2, can be isolated and manipulated (e.g., amplified, cloned, synthesized, etc.) using standard molecular biology techniques and the sequence information in the database records described herein, (e.g., described in Sambrook et al., ed., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
  • nucleic acid molecule of the invention can comprise only a portion of a nucleic acid sequence, wherein the full length nucleic acid sequence comprises a predictive marker of the invention or which encodes a polypeptide corresponding to a marker of the invention.
  • nucleic acids can be used, for example, as a probe or primer.
  • the probe/primer typically is used as one or more substantially purified oligonucleotides.
  • the oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions, e.g., hybridize under conditions of hybridization of 6X SSC at 45°C and washing in 0.2 X SSC, 0.1% SDS at 65°C, to at least about 7, preferably about 15, more preferably about 25, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, or 400 or more consecutive nucleotides of a nucleic acid of the invention.
  • stringent conditions e.g., hybridize under conditions of hybridization of 6X SSC at 45°C and washing in 0.2 X SSC, 0.1% SDS at 65°C, to at least about 7, preferably about 15, more preferably about 25, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, or 400 or more consecutive nucleotides of a nucleic acid of the invention.
  • Probes based on the sequence of a nucleic acid molecule of the invention can be used to detect transcripts or genomic sequences corresponding to one or more predictive markers of the invention.
  • the probe comprises a label group attached thereto, e.g., a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.
  • Such probes can be used as part of a diagnostic test kit for identifying cells or tissues which express the protein, such as by measuring levels of a nucleic acid molecule encoding the protein in a sample of cells from a subject, e.g., detecting mRNA levels or determining whether a gene encoding the protein has been mutated or deleted.
  • DNA sequence polymorphisms that lead to changes in the amino acid sequence can exist within a population (e.g., the human population). Such genetic polymorphisms can exist among individuals within a population due to naturally occuring allelic variation. An allele is one of a group of genes which occur alternatively at a given genetic locus. In addition, it will be appreciated that DNA polymorphisms that affect RNA expression levels can also exist that may affect the overall expression level of that gene ( e.g., by affecting regulation or degradation).
  • the terms “gene” and “recombinant gene” refer to nucleic acid molecules comprising an open reading frame encoding a polypeptide corresponding to a marker of the invention, including, e.g., sequences which differ, due to degeneracy of the genetic code, from the nucleotide sequence of nucleic acids encoding a protein which corresponds to a marker of the invention, and thus encode the same protein.
  • allelic variant refers to a nucleotide sequence which occurs at a given locus or to a polypeptide encoded by the nucleotide sequence.
  • Such naturally occuring allelic variations can typically result in 1-5% variance in the nucleotide sequence of a given gene.
  • Alternative alleles can be identified by sequencing the gene of interest in a number of different individuals. This can be readily carried out by using hybridization probes to identify the same genetic locus in a variety of individuals. Any and all such nucleotide variations and resulting amino acid polymorphisms or variations that are the result of naturally occurring allelic variation and that do not alter the functional activity are intended to be within the scope of the invention.
  • the present invention encompasses antisense nucleic acid molecules, i.e., molecules which are complementary to a sense nucleic acid of the invention, e.g., complementary to the coding strand of a double-stranded cDNA molecule corresponding to a marker of the invention or complementary to an mRNA sequence corresponding to a marker of the invention.
  • an antisense nucleic acid of the invention can hydrogen bond to (Le. anneal with) a sense nucleic acid of the invention.
  • the antisense nucleic acid can be complementary to an entire coding strand, or to only a portion thereof, e.g., all or part of the protein coding region (or open reading frame).
  • An antisense nucleic acid molecule can also be antisense to all or part of a non- coding region of the coding strand of a nucleotide sequence encoding a polypeptide of the invention.
  • the non-coding regions (“5' and 3' untranslated regions") are the 5' and 3 * sequences which flank the coding region and are not translated into amino acids.
  • An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30,
  • An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.
  • an antisense nucleic acid e.g., an antisense oligonucleotide
  • an antisense nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.
  • modified nucleotides which can be used to generate the antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5- chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1- methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguan ⁇ ne, 3-methylcytosine, 5- methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5- methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, S'-
  • the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been sub-cloned in an antisense orientation ⁇ i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
  • the nucleic acid molecules of the invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule.
  • the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids (see Hyrup et al., 1996, Bioorganic & Medicinal Chemistry 4(1): 5-23).
  • peptide nucleic acids refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained.
  • the neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength.
  • the synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup et al. (1996), supra; Perry- O'Keefe et al. (1996) Proc. Natl. Acad. ScL USA 93:14670-675.
  • PNAs can be used in therapeutic and diagnostic applications. For example,
  • PNAs can be used, e.g., in the analysis of single base pair mutations in a gene by, e.g., PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., Sl nucleases (Hyrup (1996), supra; or as probes or primers for DNA sequence and hybridization (Hyrup, 1996, supra; Perry-O'Keefe et al, 1996, Proc. Natl. Acad. ScL USA 93:14670-675).
  • PNAs can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA- DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art.
  • PNA-DNA chimeras can be generated which can combine the advantageous properties of PNA and DNA.
  • Such chimeras allow DNA recognition enzymes, e.g., RNASE H and DNA polymerases, to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity.
  • PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup, 1996, supra).
  • the synthesis of PNA-DNA chimeras can be performed as described in Hyrup (1996), supra, and Finn et al. (1996) Nucleic Acids Res. 24(17):3357-63.
  • a DNA chain can be synthesized on a solid support using standard phosphoratnidite coupling chemistry and modified nucleoside analogs.
  • the oligonucleotide can include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al, 1989, Proc. Natl. Acad. ScL USA 86:6553-6556; Lemaitre et al, 1987, Proc. Natl. Acad. ScL USA 84:648-652; PCT Publication No. WO 88/09810) or the blood-brain barrier (see, e.g., PCT Publication No. WO 89/10134).
  • peptides e.g., for targeting host cell receptors in vivo
  • agents facilitating transport across the cell membrane see, e.g., Letsinger et al, 1989, Proc. Natl. Acad. ScL USA 86:6553-6556; Lemaitre et al, 1987, Proc. Natl. Acad.
  • oligonucleotides can be modified with hybridization-triggered cleavage agents (see, e.g., Krol et al., 1988, Bio/Techniques 6:958-976) or intercalating agents (see, e.g., Zon, 1988, Pharm. Res. 5:539-549).
  • the oligonucleotide can be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.
  • the invention also includes molecular beacon nucleic acids having at least one region which is complementary to a marker of the invention, such that the molecular beacon is useful for quantitating the presence of the predictive marker of the invention in a sample.
  • a "molecular beacon" nucleic acid is a nucleic acid comprising a pair of complementary regions and having a fluorophor ⁇ and a fluorescent quencher associated therewith. The fluorophore and quencher are associated with different portions of the nucleic acid in such an orientation that when the complementary regions are annealed with one another, fluorescence of the fluorophore is quenched by the quencher.
  • Vectors including expression vectors, containing a nucleic acid encoding a polypeptide corresponding to a predictive marker of the invention can be used for production of nucleic acid and proteins corresponding to predictive markers of the invention; as well as for production of compositions relating to the predictive markers.
  • Useful vectors further comprise promoter and/or regulatory sequences for effective expression of the nucleic acid and/or protein corresponding to the predictive marker of interest.
  • promoters can include constitutive promoter/regulatory sequences, inducible promoter/regulatory sequences, tissue specific promoter/regulatory sequences, or the naturally occuring endogenous promoter/regulatory sequences corresponding to the predictive marker of interest, as required.
  • Various expression vectors are well known in the art and can be adapted to suit the particular system for expression.
  • recombinant expression vectors of the invention can be designed for expression of a polypeptide corresponding to a marker of the invention in prokaryotic (e.g., E. coli) or eukaryotic cells (e.g., insect cells ⁇ using baculovirus expression vectors ⁇ , yeast cells or mammalian cells).
  • Suitable host cells are known in the art and include those discussed in Goeddel, supra.
  • the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
  • Vectors and host cells can be produced using routine methodology known in the art.
  • use of vectors and host cells can be utilized for production of nucleic acids, polypeptides and fragments thereof corresponding to markers of the invention.
  • One aspect of the invention pertains to isolated proteins which correspond to predictive markers of the invention, and biologically active portions thereof, as well as polypeptide fragments suitable for use as immunogens to raise antibodies directed against a polypeptide corresponding to a predictive marker of the invention.
  • Polypeptides for use in the invention can be isolated, purified, or produced using the gene identification information provided herein in combination with routine molecular biology, protein purification and recombinant DNA techniques well known in the art.
  • Preferred polypeptides have the amino acid sequence listed in the one of the amino acids
  • Other useful proteins are substantially identical (e.g., at least about 70%, preferably 80%, 90%, 95%, or 99%) to one of these sequences and retain the functional activity of the protein of the corresponding naturally-occurring protein yet differ in amino acid sequence due to natural allelic variation or mutagenesis.
  • the determination of percent identity between two sequences can be accomplished using a mathematical algorithm determining the number of identical positions shared between two sequences. Determination can be carried out using any known method in the art for comparison of identity and similarity. Examples of methods used can include for example, a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci.
  • Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402.
  • PSI-Blast can be used to perform an iterated search which detects distant relationships between molecules.
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • NCBI National Center for Biotechnology Information
  • Another example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, (1988) CABIOS 4:11-17.
  • ALIGN program version 2.0
  • a PAM 120 weight residue table version 2.0
  • a gap length penalty of 12 can be used.
  • a gap penalty of 4 can be used.
  • Yet another useful algorithm for identifying regions of local sequence similarity and alignment is the FASTA algorithm as described in Pearson and Lipman (1988) Proc. Natl. Acad. ScL USA 85:2444-2448.
  • a PAM120 weight residue table can, for example, be used with a ⁇ -tuple value of 2.
  • the invention also provides chimeric or fusion proteins corresponding to a marker of the invention.
  • a "chimeric protein” or “fusion protein” comprises all or part (preferably a biologically active part) of a polypeptide corresponding to a marker of the invention operably linked to a heterologous polypeptide (i.e., a polypeptide other than the polypeptide corresponding to the marker).
  • a heterologous polypeptide i.e., a polypeptide other than the polypeptide corresponding to the marker.
  • the term "operably linked” is intended to indicate that the polypeptide of the invention and the heterologous polypeptide are fused in-frame to each other.
  • the heterologous polypeptide can be fused to the amino-terminus or the carboxyl-terminus of the polypeptide of the invention.
  • Useful fusion proteins can include a His ⁇ tag, a FLAG tag, a c-myc tag, glutathione-S-transferase (GST) tag, a hemagglutinin (HA) tag, a phage T7 gene 10 tag, a V5 tag, an herpes simplex virus (HSV) tag, and a vesicular stomatitis virus (VSV)-G tag, and any other well known heterologous tag for use in fusion protein production.
  • GST glutathione-S-transferase
  • HA hemagglutinin
  • phage T7 gene 10 tag a V5 tag
  • HSV herpes simplex virus
  • VSV vesicular stomatitis virus
  • fusion proteins can include a signal sequence from another protein such as gp67, melittin, human placental alkaline phosphatase, and phoA.
  • the fusion protein is an immunoglobulin fusion protein in which all or part of a polypeptide corresponding to a predictive marker of the invention is fused to sequences derived from a member of the immunoglobulin protein family.
  • the immunoglobulin fusion proteins of the invention can be used as immunogens to produce antibodies directed against a polypeptide of the invention in a subject, to purify ligands and in screening assays to identify molecules which inhibit the interaction of receptors with ligands.
  • An isolated polypeptide corresponding to a predictive marker of the invention, or a fragment thereof, can be used as an immunogen to generate antibodies using standard techniques for polyclonal and monoclonal antibody preparation.
  • an immunogen typically is used to prepare antibodies by immunizing a suitable (i.e. immunocompetent) subject such as a rabbit, goat, mouse, or other mammal or vertebrate.
  • An appropriate immunogenic preparation can contain, for example, recombinantly-expressed or chemically-synthesized polypeptide.
  • the preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or a similar immunostimulatory agent.
  • antibody and “antibody substance” as used interchangeably herein refer to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, Le., molecules that contain an antigen binding site which specifically binds an antigen, such as a polypeptide of the invention, e.g., an epitope of a polypeptide of the invention.
  • a molecule which specifically binds to a given polypeptide of the invention is a molecule which binds the polypeptide, but does not substantially bind other molecules in a sample, e.g., a biological sample, which naturally contains the polypeptide.
  • immunologically active portions of immunoglobulin molecules include F(ab) and F(ab') 2 fragments which can be generated by treating the antibody with an enzyme such as pepsin.
  • the invention provides polyclonal and monoclonal antibodies. Synthetic and genetically engineered variants (See U.S. Pat. No. 6,331,415) of any of the foregoing are also contemplated by the present invention. Polyclonal and monoclonal antibodies can be produced by a variety of techniques, including conventional murine monoclonal antibody methodology e.g., the standard somatic cell hybridization technique of Kohler and Milstein, Nature 256: 495 (1975) the human B cell hybridoma technique (see Kozbor et ah, 1983, Immunol.
  • the antibodies are monoclonal antibodies.
  • the antibodies of the present invention are preferably human or humanized antibodies.
  • Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supernatants for antibodies that bind the polypeptide of interest, e.g., using a standard ELISA assay.
  • the antibody molecules can be harvested or isolated from the subject (e.g., from the blood or serum of the subject) and further purified by well-known techniques, such as protein A chromatography to obtain the IgG fraction.
  • antibodies specific for a protein or polypeptide of the invention can be selected or (e.g., partially purified) or purified by, e.g., affinity chromatography to obtain substantially purified and purified antibody.
  • a substantially purified antibody composition is meant, in this context, that the antibody sample contains at most only 30% (by dry weight) of contaminating antibodies directed against epitopes other than those of the desired protein or polypeptide of the invention, and preferably at most 20%, yet more preferably at most 10%, and most preferably at most 5% (by dry weight) of the sample is contaminating antibodies.
  • a purified antibody composition means that at least 99% of the antibodies in the composition are directed against the desired protein or polypeptide of the invention.
  • recombinant antibodies such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention.
  • a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those . having a variable region derived from a murine mAb and a human immunoglobulin constant region.
  • Humanized antibodies are antibody molecules from non-human species having one or more complementarily determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule.
  • CDRs complementarily determining regions
  • Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in PCT Publication No. WO 87/02671; European Patent Application 184,187; European Patent Application 171,496; European Patent Application 173,494; PCT Publication No.
  • transgenic animals contain a substantial portion of the human antibody producing genome inserted into their own genome and the animal's own endogenous antibody production is rendered deficient in the production of antibodies.
  • Methods for making such transgenic animals are known in the art. Such transgenic animals can be made using XENOMOUSE TM technology or by using a "minilocus” approach. Methods for making XENOMICETM are described in U.S. Pat. Nos. 6,162,963, 6,150,584, 6,114,598 and 6,075,181, which are incorporated herein by reference. Methods for making transgenic animals using the "minilocus” approach are described in U.S. Pat. Nos. 5,545,807, 5,545,806 and 5,625,825; also see International Publication No. WO93/12227, which are each incorporated herein by reference.
  • Antibody fragments may be derived from any of the antibodies described above.
  • antigen-binding fragments as well as full-length monomeric, dimeric or trimeric polypeptides derived from the above-described antibodies are themselves useful.
  • Useful antibody homologs of this type include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHl domains; (ii) a F(ab'>2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHl domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., Nature 341:544-546 (1989)), which consists of a VH domain; (vii) a single domain functional heavy chain antibody, which consists of a VHH domain
  • an isolated complementarity determining region e.g., one or more isolated CDRs together with sufficient framework to provide an antigen binding fragment.
  • VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g.. Bird et al. Science 242:423-426 (1988); and Huston et al. Proc. Natl. Acad.
  • Antibody fragments such as Fv, F(ab') 2 and Fab may be prepared by cleavage of the intact protein, e.g. by protease or chemical cleavage.
  • An antibody directed against a polypeptide corresponding to a predictive marker of the invention can be used to detect the predictive marker (e.g., in a cellular sample) in order to evaluate the level and pattern of expression of the predictive marker.
  • the antibodies can also be used diagnostically to monitor protein levels in tissues or body fluids (e.g. in an tumor sample) as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include I, I, S or H.
  • the invention provides substantially purified
  • the substantially purified antibodies of the invention, or fragments thereof can be human, non-human, chimeric and/or humanized antibodies.
  • the invention provides non-human antibodies or fragments thereof, which antibodies or fragments specifically bind to a polypeptide comprising an amino acid sequence which is encoded by a nucleic acid molecule of a predictive marker of the invention.
  • Such non-human antibodies can be goat, mouse, sheep, horse, chicken, rabbit, or rat antibodies.
  • the non-human antibodies of the invention can be chimeric and/or humanized antibodies.
  • the non-human antibodies of the invention can be polyclonal antibodies or monoclonal antibodies.
  • the invention provides monoclonal antibodies or antigen binding fragments thereof, which antibodies or fragments specifically bind to a polypeptide comprising an amino acid sequence selected from the group consisting of the amino acid sequences of the present invention, an amino acid sequence encoded by the cDNA of the present invention, a fragment of at least 8, 10, 12, 15, 20 or 25 amino acid residues of an amino acid sequence of the present invention, an amino acid sequence which is at least 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence of the present invention (wherein the percent identity is determined using the ALIGN program of the GCG software package with a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4) and an amino acid sequence which is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule consisting of the nucleic acid molecules of the present invention, or a complement thereof, under conditions of hybridization of 6X SSC at 45 0 C and washing in 0.2
  • the monoclonal antibodies can be human, humanized, chimeric and/or non-human antibodies.
  • the substantially purified antibodies or fragments thereof may specifically bind to a signal peptide, a secreted sequence, an extracellular domain, a transmembrane or a cytoplasmic domain or cytoplasmic membrane of a polypeptide of the invention.
  • the substantially purified antibodies or fragments thereof, the non-human antibodies or fragments thereof, and/or the monoclonal antibodies or fragments thereof, of the invention specifically bind to a secreted sequence or an extracellular domain of the amino acid sequences of the present invention.
  • the invention also provides a kit containing an antibody of the invention conjugated to a detectable substance, and instructions for use.
  • Still another aspect of the invention is a diagnostic composition comprising an antibody of the invention and a pharmaceutically acceptable carrier.
  • the diagnostic composition contains an antibody of the invention, a detectable moiety, and a pharmaceutically acceptable carrier.
  • a sample of cancerous cells is obtained from a patient.
  • An expression level is measured in the sample for a marker corresponding to at least one of the predictive markers set forth in Table 1 and Table 2.
  • a marker set is utilized comprising markers identified in Table 1 and/or Table 2, and put together in a marker set using the methods described herein. Such analysis is used to obtain an expression profile of the tumor in the patient.
  • Evaluation of the expression profile is then used to determine whether the patient is a long term survivor and would benefit from proteasome inhibition therapy (e.g., treatment with a proteasome inhibitor (e.g., bortezomib) alone, or in combination with additional agents) and/or glucocorticoid therapy (e.g., treatment with a glucocorticoid (e.g., dexamethasone) alone, or in combination with additional agents), or an alternative agent expected to have a similar effect on survival.
  • proteasome inhibition therapy e.g., treatment with a proteasome inhibitor (e.g., bortezomib) alone, or in combination with additional agents
  • glucocorticoid therapy e.g., treatment with a glucocorticoid (e.g., dexamethasone) alone, or in combination with additional agents
  • an alternative agent expected to have a similar effect on survival e.g., treatment with a protea
  • Evaluation of the expression profile can also be used to determine whether a patient is a short term survivor and would benefit from a cancer therapy other than proteasome inhibition and/or glucocorticoid therapy or would benefit from an altered proteasome inhibition therapy regimen and/or glucocorticoid therapy regimen.
  • Evaluation can include use of one marker set prepared using any of the methods provided or other similar scoring methods known in the art (e.g., weighted voting, combination of threshold features (CTF), Cox proportional hazards analysis, principal components scoring, linear predictive score, K-nearest neighbor, etc), e.g., using expression values deposited with the Gene Expresion Omnibus (GEO) program at the National Center for Biotechnology Information (NCBI, Bethesda, MD).
  • GEO Gene Expresion Omnibus
  • evaluation can comprise use of more than one prepared marker set.
  • a proteasome inhibition therapy and/or glucocorticoid therapy will be identified as appropriate to treat the cancer when the outcome of the evaluation demonstrates a long term survivor or a more aggressive therapy regimen will be identified for a short term survivor.
  • the invention features a method of evaluating a patient, e.g., a patient with cancer, e.g. a hematological cancer (e.g., multiple myeloma, leukemias, lymphoma, etc) or cancer from a solid tumor (e.g., in lung, breast, prostate, ovary, colon, kidney, or liver) for short term or long term survival.
  • a hematological cancer e.g., multiple myeloma, leukemias, lymphoma, etc
  • a solid tumor e.g., in lung, breast, prostate, ovary, colon, kidney, or liver
  • the method includes providing an evaluation of the expression of the markers in a predictive marker set of markers in the patient, wherein the predictive marker set has the following properties: it includes a plurality of genes, each of which is differentially expressed as between patients short term and long term survivor patients and non-afflicted subjects and it contains a sufficient number of differentially expressed markers such that differential expression (e.g., as compared to a level in a non-afflicted reference sample) of each of the markers in the predictive marker set in a subject is predictive of short term or long term survival with no more than about 15%, about 10%, about 5%, about 2.5%, or about 1% false positives (wherein false positive means predicting that a patient as responsive or non-responsive when the subject is not); and providing a comparison of the expression of each of the markers in the set from the patient with a reference value, thereby evaluating the patient.
  • the predictive marker set has the following properties: it includes a plurality of genes, each of which is differentially expressed as between patients short term and long term survivor patients and
  • the cancer may have become resistant to proteasome inhibition therapy and/or glucocorticoid therapy, and another treatment protocol should be initiated to treat the patient.
  • these determinations can be made on a patient by patient basis or on an agent by agent (or combinations of agents). Thus, one can determine whether or not a particular proteasome inhibition therapy and/or glucocorticoid therapy is likely to benefit a particular patient or group/class of patients, or whether a particular treatment should be continued.
  • information e.g., about the patient's marker expression levels
  • a patient is expected to be a short term or long term survivor
  • a third party e.g., a hospital, clinic, a government entity, reimbursing party or insurance company (e.g., a life insurance company).
  • a third party e.g., a hospital, clinic, a government entity, reimbursing party or insurance company (e.g., a life insurance company).
  • a third party e.g., a hospital, clinic, a government entity, reimbursing party or insurance company (e.g., a life insurance company).
  • choice of medical procedure, payment for a medical procedure, payment by a reimbursing party, or cost for a service or insurance can be function of the information.
  • the third party receives the information, makes a determination based at least in part on the information, and optionally communicates the information or makes a choice of procedure, payment, level of payment, coverage, etc. based on the information.
  • informative expression level of a predictive marker or a predictive marker set selected from or derived from Table 1 and/or Table 2 is determined.
  • a premium for insurance (e.g., life or medical) is evaluated as a function of information about one or more marker expression levels, e.g., a predictive marker or predictive marker set, e.g., a level of expression associated with short term or long term survival (e.g., the informative expression level).
  • a predictive marker or predictive marker set e.g., a level of expression associated with short term or long term survival (e.g., the informative expression level).
  • premiums can be increased (e.g., by a certain percentage) if the markers of a patient or a patient's predictive marker set described herein are differentially expressed between an insured candidate (or a candidate seeking insurance coverage) and a reference value (e.g., a non-afflicted person).
  • Premiums can also be scaled depending on marker expression levels, e.g., the result of evaluating a predictive marker or predictive marker set described herein. For example, premiums can be assessed to distribute risk, e.g., as a function of marker expression levels, e.g., the result of evaluating a predictive marker or predictive marker set described herein. In another example, premiums are assessed as a function of actuarial data that is obtained from patients that are short term or long term survivors.
  • Information about marker expression levels can be used, e.g., in an underwriting process for life insurance.
  • the information can be incorporated into a profile about a subject. Other information in the profile can include, for example, date of birth, gender, marital status, banking information, credit information, children, and so forth.
  • An insurance policy can be recommended as a function of the information on marker expression levels, e.g., the result of evaluating a predictive marker or predictive marker set described herein, along with one or more other items of information in the profile.
  • An insurance premium or risk assessment can also be evaluated as function of the predictive marker or predictive marker set information.
  • points are assigned on the basis of being a short term or long term survivor.
  • information about marker expression levels is analyzed by a function that determines whether to authorize the transfer of funds to pay for a service or treatment provided to a subject (or make another decision referred to herein).
  • a function that determines whether to authorize the transfer of funds to pay for a service or treatment provided to a subject (or make another decision referred to herein).
  • the results of analyzing a expression of a predictive marker or predictive marker set described herein may indicate that a subject is a short term or long term survivor, suggesting that a treatment course is needed, thereby triggering an outcome that indicates or causes authorization to pay for a service or treatment provided to a subject.
  • informative expression level of a predictive marker or a predictive marker set selected from or derived from Table 1 and/or Table 2 is determined and payment is authorized if the informative expression level identifies a long term survivor.
  • an entity e.g., a hospital, care giver, government entity, or an insurance company or other entity which pays for, or reimburses medical expenses, can use the outcome of a method described herein to determine whether a party, e.g., a party other than the subject patient, will pay for services (e.g., a particular therapy) or treatment provided to the patient.
  • a first entity e.g., an insurance company
  • a first entity e.g., an insurance company
  • the disclosure features a method of providing data.
  • the method includes providing data described herein, e.g., generated by a method described herein, to provide a record, e.g., a record described herein, for determining if a payment will be provided.
  • the data is provided by computer, compact disc, telephone, facsimile, email, or letter, ⁇ i some embodiments, the data is provided by a first party to a second party.
  • the first party is selected from the subject, a healthcare provider, a treating physician, a health maintenance organization (HMO), a hospital, a governmental entity, or an entity which sells or supplies the drug.
  • HMO health maintenance organization
  • the second party is a third party payor, an insurance company, employer, employer sponsored health plan, HMO, or governmental entity.
  • the first party is selected from the subject, a healthcare provider, a treating physician, an HMO, a hospital, an insurance company, or an entity which sells or supplies the drug and the second party is a governmental entity.
  • the first party is selected from the subject, a healthcare provider, a treating physician, an HMO, a hospital, an insurance company, or an entity which sells or supplies the drug and the second party is an insurance company.
  • the disclosure features a record (e.g., computer readable record) which includes a list and value of expression for the predictive marker or predictive marker set for a patient.
  • the record includes more than one value for each marker.
  • Phase 2 myeloma studies were conducted in order to allow a more precise estimate of anti-tumor activity of bortezomib in a more homogeneous population of patients.
  • the safety and efficacy of bortezomib in subjects with multiple myeloma was investigated in two phase 2 clinical studies, M34100-024 (subjects with first relapse) and M34100-025 (subjects with second or greater relapse and refractory to their last prior therapy).
  • a multicenter, open-label, randomized study was conducted, comprising 627 enrolled patients with relapsed or refractory multiple myeloma (Protocol M34101-039). See Richardson et.al., N.Engl. J. Med,. 352:2487-2498 (2005). Patients were treated with either bortezomib (315 patients) or high-dose dexamethasone (312 patients).
  • a sterile lyophilized powder for reconstitution was supplied in vials containing 2.5 mg bortezomib and 25 mg mannitol USP.
  • Each vial was reconstituted with 2.5 mL of normal (0.9%) saline, Sodium Chloride Injection USP, such that the reconstituted solution contained bortezomib at a concentration of 1 mg/mL.
  • the reconstituted solution was clear and colorless with a final pH between 5 and 6.
  • Randomization was to be stratified, based on the number of lines of prior therapy (one prior line versus more than one prior line of therapy), time of progression relative to treatment (progression while on their most recent therapy or within 6 months of stopping their most recent therapy, or relapse >6 months after receiving their most recent therapy), and screening ⁇ 2 -microglobulin levels (>2.5 mg/L versus ⁇ 2.5 mg/L).
  • Patients assigned to the bortezomib group received treatment for a maximum of 273 days. Patients in this treatment group received up to eight 3-week treatment cycles followed by up to three 5-week treatment cycles of bortezomib. Within each 3-week treatment cycle, the patient received bortezomib 1.3 mg/m 2 /dose alone as a bolus intravenous (IV) injection twice weekly for two weeks (on Days 1, 4, 8, and 11) of a 21 -day cycle. Within each 5-week treatment cycle, the patient received bortezomib 1.3 mg/m 2 /dose alone as a bolus IV injection once weekly (on Days 1, 8, 15, and 22) of a 35-day cycle.
  • IV intravenous
  • Patients assigned to the high-dose dexamethasone group received treatment for a maximum of 280 days. Patients in this treatment group received up to four 5-week treatment cycles, followed by up to five 4-week treatment cycles. Within each 5-week treatment cycle, the patient received dexamethasone 40 mg/day PO, once daily on Days 1 to 4, 9 to 12, and 17 to 20 of a 35-day cycle. Within each 4-week treatment cycle, the patient received dexamethasone 40 mg/day PO once daily on Days 1 to 4 of a 28 day cycle.
  • the protocol provided for patients in the dexamethasone group who experienced confirmed progressive disease (PD) to receive bortezomib on a companion study (An International, Non-Comparative, Open-Label Study of PS-341 Administered to Patients with Multiple Myeloma Who Received High-dose Dexamethasone or Experienced Progressive Disease after Receiving at Least Four Previous Therapies, (Protocol M34101-040).
  • An additional 240 patients who did not participate in this study, enrolled in the companion study and according to the protocol would have received at least four prior therapies. Pharmacogenomic samples were also sought for these 240 patients.
  • the response rate (complete plus partial response(CR + PR)) in the bortezomib group was 38 percent; and in the dexamethasone group was 18 percent (P ⁇ 0.0001).
  • Complete response was achieved in 20 patients (6 percent) who received bortezomib, and in 2 patients ( ⁇ 1 percent) who received dexamethasone (P ⁇ 0.001),'with complete response plus near-complete response in 13 and 2 percent (P ⁇ 0.0001) in patients receiving bortezomib and dexamethasone, respectively.
  • Pharmacogenomic samples collected [00195] Pharmacogenomic tumor samples (bone marrow aspirate) were collected from patients for evaluation of the expression of global mRNA levels.
  • the level of expression of predictive markers in any bortezomib study alone, or in combination, can be used to develop classifiers for prediction of short or long term survival after proteasome inhibition therapy, using statistical methods known in the art.
  • the level of expression of markers in the -039 dexamethasone study can be used to develop classifiers for short or long term survival after glucocorticoid therapy.
  • Biopsies from 264 multiple myeloma patients with survival information resulted in generation of high quality gene expression data which was used to identify predictive markers.
  • Candidate markers that are associated with the survival of multiple myeloma patients receiving proteasome inhibition (e.g., bortezomib) therapy or glucocorticoid (e.g., dexamethasone) therapy were selected by using Cox proportional hazards modeling.
  • the myeloma cells were enriched via rapid negative selection ( Figure IA).
  • the enrichment procedure employs a cocktail of cell-type specific antibodies coupled with an antibody that binds red blood cells RosetteSep (Stem Cell Technologies).
  • the antibody cocktail has antibodies with the following specificity: CD 14 (monocytes), CD2 (T and NK cells), CD33 (myeloid progenitors and monocytes), CD41 (platelets and megakaryocytes), CD45RA (na ⁇ ve B and T cells) and CD66b (granulocytes).
  • the antibodies cross-linked the non-myeloma cell types to the red blood cells in the samples.
  • the bound cell types were removed using a modified ficoll density gradient.
  • RNA (if available) was converted to biotinylated cRNA by a standard T7 based amplification protocol (AFFYMETRK® Inc., Santa Clara, CA). A small number of samples with >0.5 — 2.0 ⁇ g were also labeled and subsequently hybridized if 6 ⁇ g of cRNA was produced.
  • Samples from clinical trials 025 and 040 were randomized by clinical site and operator, assigned to batches of 24 samples and labeled by manual T7 amplification (Batchl). Samples from clinical trial 039 were randomized by clinical site and assigned to 95 sample batches and labeled by an automated T7 amplification procedure (Batch 2).
  • cDNA and the biotin labeled cRNA were purified using AMPURE® PCR Purification System, following the manufacturer's protocol (AGENCOURT® Bioscience Corporation, Beverly, MA).
  • the cRNA yield was assessed by spectrophotometry and 10 ⁇ g of cRNA was fragmented and further processed for triplicate hybridization on the AFFYMETRIX® Human Genome HG-U133A and HG-U133B GENECHIP® arrays. In cases where cRNA yield ranged between 6 ⁇ g to 10 ⁇ g, the entire cRNA sample was fragmented.
  • cRNA for each sample was hybridized to the U133A/B arrays in triplicate; operators, chip lots, clinical sites and scanners (GENECHIP® Scanner 3000) were controlled throughout. Background subtraction, smoothing adjustment, noise corrections, and signal calculations were performed with AFFYMETRIX® MAS5.0. Quality control metrics determined by AFFYMETRIX® analysis and MPI included: percent present call (>25) scale factor ( ⁇ 11), ⁇ - actin 3':5' ratio ( ⁇ 15) and background ( ⁇ 120). Samples that fell outside these metrics were excluded from subsequent analysis.
  • the myeloma purity score examines expression of genes known in the literature to be expressed highly in myeloma cells (and their normal plasma precursor cells), to expression of genes known to be expressed highly in erythroid cells, neutrophils and T cells - see list of 14 markers below).
  • the myeloma score expression of myeloma markers (#1-4 below) / erythroid (#5-7) + neutrophil (#8-11) + T cell (#12-14 below):
  • Myeloma purity scores of representative samples are illustrated in Figure IB. Samples with a myeloma purity score less than 10 were excluded from further analysis. Normalization and Logarithmic Transformation. [00206] Expression values for all markers on each microarray were normalized to a trimmed mean of 150. Expression values were determined using MAS5 gene expression analysis data processing software (AFFYMETRDC® Inc., Santa Clara, CA). These values will be referred to as the "normalized expression" in the remainder of this section. In a further processing step, the median expression level was determined across repeated expression measurements for the same sample.
  • the median expression level values for the markers are being submitted to the Gene Expression Omnibus (GEO) program, a gene expression/molecular abundance repository, at the National Center for Biotechnology Information (NCBI, Bethesda, MD) and will be searchable and retrievable at the NCBI website. These median expression level values are incorporated herein by reference. The logarithm base 2 was taken of the resulting median expression level, and this value will be referred to as the "log expression” in the remainder of this section. Variance Components Analysis.
  • GEO Gene Expression Omnibus
  • the probe sets were reduced in number to include only those having more than 65% of their variance due to patient sample variance. Of 44,928 probe sets, 9,200 passed this filter and were carried on to further analysis.
  • Single gene transcripts that are associated with patient survival can be identified using the survival analysis methodology described below.
  • Predictive markers identified using the methodology described herein are set forth in Table 1 and Table 2.
  • Table 1 markers were identified using bortezomib-treated patients across studies 025, 039 and 040.
  • Table 2 markers were identified using patients in studies 025 and 040, and were used to predict survival outcome in 039, as shown in Figure 3 and described below.
  • a set of one or more gene transcripts that together classify samples into short term and long term survivors, in the context of a particular classifier algorithm, is referred to as a
  • model The gene transcripts are referred to as “features.” Determining which combination of gene transcript(s) best classifies samples into sensitive and resistant groups is referred to as "model selection.” The following section describes the process of how the models of the present invention were identified.
  • the methods provided herein along with the single marker identification or predictive markers can be used to identify additional models comprising markers of the invention.
  • classification methods for building models to classify samples based on their features can be used with the markers in Table 1 or 2 to build predictive models. For example, predictors are based on linear combinations of the expression values (Golub et al., Science, 286:531-7 (1999), Radmacher et al. J. Comput. Biol., 9:505-12 (2002)).
  • predictors are based on neural networks, which can be used to predict survival time directly or to develop reduced dimensionality representations of the expression data which can then be fed into a Cox proportional hazards model (Khan et al., Nat. Med. 7:673-9 (2001), Nguyen et al., Bioinformatics 18:39-50 (2002), Lundina et al., Oncology 57:281-286 (1999)).
  • There are many other methods for defining a multivariate predictor, all of which can be adapted to use with survival data (Ripley BD. Pattern recognition and neural networks (Cambridge (U.K.): Cambridge University Press; (1996), Dudoit et al., J Am. Stat. Assoc. 97:77-87 (2002)). These can be used for survival by threshold the survival time to turn it into a classification problem (low- and high-risk)
  • the first step in predictive model selection is to filter the 9,200 features down to a smaller number which show a correspondence with the sample classifications. Filtering involves first ranking the features by a scoring method, and then taking only the highest ranking features for further analysis.
  • the filtering algorithm used in the present invention was Cox proportional hazards modeling to determine a /?-value for the association of a feature with time to progression and death.
  • a Cox proportional hazard analysis was performed to determine predictors of time until death in patients with relapsed and refractory multiple myeloma after treatment. This methodology is designed to analyze time to event data where some of the data may be censored (see E.T. Lee, Statistical Methods for Survival Data Analysis, 2 nd ed. 1992, John Wiley& Sons, Inc.).
  • the p-value of the Cox proporitional hazards model built on the transformed test data indicates whether the selected genes have predictive value.
  • patients can be divided into low- and high-risk groups.
  • a log-rank test is applied to the outcome data of these groups of patients to determine whether the difference between the predicted high- and low-risk groups is significant.
  • a classifier was developed from analysis of the level of expression of the markers in the -025 and the -040 studies using the Principal Components algorithm. The expression levels in samples from these studies were combined to build a model using the 100 probesets with strongest superpc scores listed in column 2 of Table 2. This set of probes (Table 2; "survival classifier”) was applied as a training set to the -039 studies.
  • 025+040 model as the threshold on superpc score was varied. The ability to distinguish long term survivors from short term survivors was retained throughout near all (but the smallest) models. Assignments were determined to be quite stable: most samples are assigned to the same class (long term survivor or short term survivor) regardless of the number of probesets included in the model.
  • Probeset DD corresponds to the AFFYMETRDC® Inc. (Santa Clara, CA) identifier from the Human Genome Ul 33 A, B set oligonucleotide arrays which were used;
  • Gene symbol corresponds to a symbol the gene is commonly known by, and was also taken from the AFFYMETRIX® Inc. annotation files;
  • TTP Marker or "TTP”represents indication of predictive marker which is significantly upregulated in samples with a correlation to longer time to progression (+), or are significantly upregulated in samples with a correlation to shorter time to progression (-).
  • the "V” represents bortezomib and "D” represents dexamethasone.
  • a “+” represents good prognosis for time to progression, a “-”represents a poor prognosis for time to progression;
  • Response Marker or “Resp” represents indication of predictive marker which is significantly upregulated in samples which are responsive to therapy (+), or are significantly upregulated in samples which are non-responsive to therapy (-).
  • the "V” represents bortezomib and “D” represents dexamethasone.
  • a “+” represents responsive, a "-” represents non responsive.
  • Super PC 025+040 represents the superPC score for each probeset upon analysis of expression levels in samples from the 025 and 040 studies. Probesets with positive values are associated with shorter survival time and probesets with negative values are associated with longer survival time.
  • Predictive markers of the invention are provided in Tables 1 and 2.
  • Table 1 sets forth predictive markers identified which are specific identifiers of long term and short term survival. Marker nos. 225 to 403 in Table 1 are upregulated in long term survivors; marker nos. 1 to 224 are upregulated in short term survivors.
  • Table 1 also indicates markers which are correlated with time to progression or response to a treatment (see, International Patent Publication No. WO04053066, published June 24, 2004, or U.S. Patent Application No. 11/449,195, filed June 8, 2006).
  • Table 2 also sets forth predictive markers identified which are specific identifiers of long term or short term survival. Marker nos. 38 to 100 in Table 2 are upregulated in long term survivors; marker nos.
  • the Linear Predictive Score was implemented as described by Wright et al., "A gene-expression based method to diagnose clinically distinct groups of diffuse large B cell lymphoma.” PNAS 100(17): 9991-9996 (2003), the contents of which are incorporated herein by reference. As described by Wright et al., the LPS score for a vector X is computed as :
  • X 1 represents the log expression value for the 7 th feature in the set
  • a j is a scaling factor representing the degree to which the j 01 feature is associated with the outcome to be predicted.
  • t-statistics of the features For the LPS score, the likelihood that a sample is in the first of the two classes is determined using this formula:
  • ⁇ (x; ⁇ , ⁇ 2 ) represents the normal density function with mean ⁇ and variance ⁇ 2
  • /Z 1 , ⁇ , 2 , ⁇ 2 and ⁇ 2 are the observed means and variances of the LPS scores for category 1 and category 2.
  • category 1 would be responders
  • category 2 would be non- responders.
  • the K-nearest neighbor classification method computes the similarity between a query profile and each of the profiles in the training set [Introduction to Machine Learning by Ethem ALPAYDIN, The MIT Press, October 2004, ISBN 0-262-01211-1].
  • the k most similar profiles are selected, and a vote is taken amongst their class labels to determine the prediction for the query profile.
  • k l.
  • Feature selection is the process of determining a subset of the thousands of available features in the dataset, resulting in a combination of features that form a marker set or model, to classify patients by treatment outcome. There are many approaches to selecting features.
  • Fluorescence cell sorting analysis of pre- and post-enrichment samples demonstrated that the enrichment could yield samples of 80-90% tumor cells (Fig 1). FACS analyses were not practical at all participating centers. Therefore, we assessed sample purity via analysis of a myeloma purity score derived from the microarray data (see methods) and samples with low tumor cell purity were excluded from further analyses (Fig IB). Sample attrition was observed at each step in the process of generating gene expression data. Approximately 60% of samples exhibited RNA quantity and quality adequate for hybridization. Of these samples, ⁇ 85% generated high quality microarray data and then 85% passed the assessment of tumor cell enrichment described above. These results were generally consistent across the different clinical trials.
  • genomic subset .of patients with genomic data were representative of the general trial population. No bias was observed with regard to age, gender, or myeloma isotype. For some of these trials the survival values of the genomics subset were indicative of a worse outcome. Although serum albumin and serum ⁇ -2 microglobulin were elevated in the genomics subset of the 025 trial this was not observed in the other trial data. The genomics subset of each trial, however, did exhibit a higher baseline tumor burden in the bone marrow aspirate, indicating that successful sampling is likely related to the extent of marrow disease. The data suggests that genomic subsets are reasonable representations of the study populations as a whole, although there is an overrepresentation of patients with high tumor burden. Comparison ofdataset with published myeloma biology
  • Hardin J, Kordsmeier B, et al Global gene expression profiling of multiple myeloma, monoclonal gammopathy of undetermined significance, and normal bone marrow plasma cells.
  • Figure 2A shows a dendrogram of 264 myeloma patient samples and 6 normal plasma cell control (PC) samples. Patients with different age, gender and myeloma isotype were randomly distributed ( Figure 2A) across these groups. Further, there was no significant clustering of samples that originated at the same clinical center.
  • the probesets comprising this survival classifier (Table 2) do not overlap with the response classifier. This is not surprising, as the survival and response endpoints are only partially related. Overexpression of adhesion related genes (CDHl, CD36, TNXB) are correlated with longer survival, and cancer antigens (SSX4, SSX2) are correlated with shorter survival, suggesting there may be biological consistencies.
  • survival classifier described here captures outcome-related information that is distinct from clinical prognostic variables (e.g. serum albumin and ⁇ -2M) as demonstrated by the significant capacity to discern risk groups within the high and low-risk ISS groups (Figure 5). Studies in lymphoma have drawn similar conclusions (Rosenwald N,Engl.J.Med. 346:1937-47 (2002).

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Abstract

La présente invention concerne l'identification de marqueurs prévisionnels qui peuvent être utilisés pour déterminer si des patients cancéreux sont supposés manifester des temps de survie à long terme ou à court terme. La présente invention concerne en particulier l'utilisation de certains marqueurs prévisionnels individuels et/ou combinés, l'expression des marqueurs prévisionnels correspondant à la survie à court terme ou à long terme escomptée. En conséquence, en examinant les niveaux d'expression de marqueurs prévisionnels individuels et/ou de marqueurs prévisionnels contenant un ensemble de marqueurs, il est possible de déterminer la survie prédite d'un patient.
PCT/US2007/017716 2006-08-10 2007-08-09 Procédés d'identification, d'évaluation, et de traitement de patients soumis à une thérapie anticancéreuse WO2008021183A2 (fr)

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CA002660275A CA2660275A1 (fr) 2006-08-10 2007-08-09 Procedes d'identification, d'evaluation, et de traitement de patients soumis a une therapie anticancereuse
JP2009523844A JP5725711B2 (ja) 2006-08-10 2007-08-09 癌治療法を有する患者の同定、評価、および治療のための方法
EP07836670A EP2046973A4 (fr) 2006-08-10 2007-08-09 Procédés d'identification, d'évaluation, et de traitement de patients soumis à une thérapie anticancéreuse
AU2007284724A AU2007284724B2 (en) 2006-08-10 2007-08-09 For the identification, assessment, and treatment of patients with cancer therapy
MX2009001489A MX2009001489A (es) 2006-08-10 2007-08-09 Para la identificacion, valoracion y tratamiento de pacientes con terapia de cancer.

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US20170166974A1 (en) * 2014-05-20 2017-06-15 Erasmus University Medical Center Rotterdam Method for the treatment of multiple myeloma
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JP6968058B2 (ja) 2015-09-24 2021-11-17 メイヨ・ファウンデーション・フォー・メディカル・エデュケーション・アンド・リサーチ 質量分析法による免疫グロブリン遊離軽鎖の同定
US20180312928A1 (en) * 2015-10-26 2018-11-01 Cipherome Method and system for selecting customized drug using genomic nucleotide sequence variation information and survival information of cancer patient
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AU2007284724A1 (en) 2008-02-21
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US9500656B2 (en) 2016-11-22
EP2046973A2 (fr) 2009-04-15
EP2046973A4 (fr) 2010-04-07
WO2008021183A3 (fr) 2009-04-02
AU2007284724B2 (en) 2014-03-13
JP5725711B2 (ja) 2015-05-27
JP2010500565A (ja) 2010-01-07
WO2008021183A2 (fr) 2008-02-21

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